CN117965608A

CN117965608A - Application of cotton high-resistance Wen Jiyin GH_D07G0997

Info

Publication number: CN117965608A
Application number: CN202410134372.7A
Authority: CN
Inventors: 梁亚军; 龚照龙; 王俊铎; 郑巨云; 阳妮; 孙丰磊; 李雪源; 耿世伟; 牛康康; 钱帅帅
Original assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute
Current assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute
Priority date: 2024-01-31
Filing date: 2024-01-31
Publication date: 2024-05-03

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of cotton high-resistance Wen Jiyin GH_D07G 0997. The invention obtains the gene GH_D07G0997 which relates to the cotton chloroplast pathway and is obviously differentially expressed in the transcriptome, and uses the VIGS technology to carry out biological function verification, and the invention discovers that the plant with the silent GH_D07G0997 gene has poor stress resistance compared with the contrast, and the plant with the over-expressed GH_D07G0997 gene has better stress resistance. GH_D07G0997 responds to high temperature stress by affecting plant water and malondialdehyde, does not participate in the proline biosynthesis pathway, and plays a positive regulation role in the high temperature stress process.

Description

Application of cotton high-resistance Wen Jiyin GH_D07G0997

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to application of cotton high-resistance Wen Jiyin GH_D07G 0997.

Background

Cotton (Gossypiumhirsutum Linn) is a seed fiber of Malvaceae (Malvaceae), cotton (Gossypium) plants, and is one of the main cash crops in China. Xinjiang has become the largest cotton production place for China, and the total yield is more than 90% of the whole country. In the cotton area of Xinjiang, the periodicity is high Wen Tianqi, especially in the cotton full bloom stage and the cotton boll setting stage of 7 and 8 months, the young buds of cotton drop off and dry, the pollen activity is reduced, the malformed bolls and stiff bolls are increased, and the cotton bolls drop off on the middle and upper fruit branches seriously, so that the boll setting rate and the ginned cotton yield are seriously affected. The high temperature has become an important problem for restricting the quality improvement and the supply of Xinjiang cotton. The breeding of high temperature resistant varieties is a fundamental measure for coping with high temperature stress, breaking the annual wandering bottleneck of single yield and coping with future global warming. However, at present, available cotton high temperature resistant genes and germplasm are fewer, and the method is particularly important for excavating high temperature resistant related genes and cultivating new cotton high temperature resistant varieties.

Disclosure of Invention

The invention aims to excavate the high temperature resistant related genes of cotton, analyze the mechanism of the cotton responding to high temperature stress, screen and create high temperature resistant cotton germplasm.

In order to achieve the above object, the present invention provides the use of Wen Jiyin gh_d07G0997, which comprises one or more of the following (a) to (c);

(a) Analyzing the mechanism of the cotton responding to the high temperature stress;

(b) Screening high temperature resistant cotton seeds;

(c) Creating high temperature resistant cotton germplasm;

the GH_D07G0997 has an accession number XP_040953438.1 at NCBI.

Preferably, the creating the high temperature resistant cotton germplasm comprises: overexpressing the cotton high temperature resistant gene to obtain a high temperature resistant cotton plant;

the cotton includes upland cotton;

The ambient temperature for the application was 40 ℃.

The invention also provides a silencing expression vector for reducing the high temperature stress resistance of cotton, which comprises a gene silencing vector skeleton and a silencing gene fragment inserted into the gene silencing vector skeleton;

the silent gene fragment is a characteristic fragment of the GH_D07G0997 gene;

The nucleotide sequence of the characteristic fragment of the GH_D07G0997 gene is shown in SEQ ID NO. 9.

Preferably, the gene silencing vector backbone comprises pTRV2.

The invention also provides a VIGS silencing system for reducing the high temperature stress resistance of cotton, which comprises an agrobacterium tumefaciens liquid containing TRV1 and the agrobacterium tumefaciens liquid containing the silencing expression vector according to the technical scheme.

Preferably, the volume ratio of the agrobacterium tumefaciens solution containing TRV1 to the agrobacterium tumefaciens solution containing the silencing expression vector is 1:1, a step of;

The OD value of the agrobacteria liquid containing TRV1 is 0.8-1.0;

The OD value of the agrobacterium tumefaciens bacterial liquid containing the silencing expression vector is 0.8-1.0.

The invention also provides a method for reducing the high temperature stress resistance of cotton, which comprises the following steps: the VIGS silencing system described in the above technical scheme is used to infect cotton plants.

Preferably, the means of infestation comprises leaf injection.

The invention also provides a recombinant vector for improving the high temperature stress resistance of cotton, which comprises a basic vector and a GH_D07G0997 gene inserted into the basic vector.

The invention also provides a method for improving the high temperature stress resistance of cotton, which comprises the following steps: the recombinant vector in the technical scheme is introduced into cotton plants.

The beneficial effects are that:

The invention combines the whole genome association analysis and ONT transcriptome sequencing technology with the phenotype character and physiological index of cotton under high temperature stress to obtain the gene GH_D07G0997 which relates to the cotton chloroplast pathway and is obviously differentially expressed in transcriptome, and uses the VIGS technology to carry out biological function verification, so that plants with silent GH_D07G0997 genes are found to have better stress resistance than the control plants. GH_D07G0997 responds to high temperature stress by affecting plant water and malondialdehyde, does not participate in the proline biosynthesis pathway, and plays a negative regulation role in the high temperature stress process.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a Manhattan diagram of a GWAS located chloroplast-associated SNP site-an inverted three-fruit branch abscission number-associated site;

FIG. 2 is a Manhattan plot of the GWAS located chloroplast-associated SNP locus-the number of dry buds associated locus;

FIG. 3 shows phenotype and NBT staining results of 40℃high temperature stressed cotton;

FIG. 4 is a graph showing the results of ROS accumulation analysis in New land early No. 36 and in vehicles 61-72 in response to high temperature stress; wherein the different lowercase letters represent mean marks differing by significance when the significance level is 0.05; * Indicating that the difference is significant, indicating that the difference is extremely significant;

FIG. 5 shows the results of fluorescence change detection of chlorophyll in New land early No. 36 and in vehicles 61-72 treated differently;

FIG. 6 is a novel gene expression profile;

FIG. 7 is a representative lncRNA and target gene analysis results;

FIG. 8 shows the differential expression level of the candidate gene GH_D07G0997 transcriptome; wherein the different lowercase letters represent mean marks differing by significance when the significance level is 0.05; r represents a temperature-sensitive variety, and T represents a temperature-resistant variety;

FIG. 9 shows the gene structure of candidate gene GH_D07G 0997;

FIG. 10 is a tertiary structure of candidate gene GH_D07G 0997;

FIG. 11 is a graph showing the observation of the albino phenotype of the TRV-CLA1 positive control;

FIG. 12 is a graph showing silencing efficacy measurements; wherein P <0.05, P <0.01;

FIG. 13 is a phenotype of GH_D07G0997 silenced plants after 40℃treatment;

FIG. 14 shows the results of physiological index measurements before and after treatment of silenced GH_D07G0997 plants at 40 ℃; wherein, the difference is significant, the difference is extremely significant;

FIG. 15 shows the PCR detection results of transgenic positive plants;

FIG. 16 is a phenotype of a plant over-expressing a gene of interest after treatment at 40 ℃.

Detailed Description

The invention provides application of cotton high-tolerance Wen Jiyin GH_D07G0997, comprising one or more of the following (a) - (c);

(b) Screening high temperature resistant cotton seeds;

(c) Creating high temperature resistant cotton germplasm;

the GH_D07G0997 has an accession number XP_040953438.1 at NCBI.

In the invention, the GH_D07G0997 gene belongs to a member of a zinc binding dehydrogenase family of proteins of an oxidoreductase, has an oxidoreductase activity, is combined with zinc ions to participate in the redox process, is positioned in chloroplast thylakoid membranes, chloroplasts, plasma membranes and vacuoles, and has a homology of AT4G13010 in Arabidopsis thaliana, and the similarity is 98%. The invention provides a method for creating high temperature resistant cotton seeds, which comprises the following steps: and overexpressing the cotton high temperature resistant gene to obtain a high temperature resistant cotton plant. The cotton of the present invention preferably comprises upland cotton; the ambient temperature of the application is preferably 40 ℃.

the silent gene fragment is a characteristic fragment of the GH_D07G0997 gene;

The nucleotide sequence of the characteristic fragment of the GH_D07G0997 gene is shown as SEQ ID NO.9, and specifically comprises the following steps: 5'-GTCAAAAGCCTCGGAGCAGACCAAGGAATGCCCATTGCAC-3'.

In the present invention, the gene silencing vector backbone preferably comprises pTRV2.

The invention constructs a silencing expression vector of the GH_D07G0997 gene, and introduces the silencing expression vector into cotton plants, and the result shows that the plant with the GH_D07G0997 gene silenced has poorer tolerance to high temperature than a control.

In the present invention, the volume ratio of the agrobacterium solution containing TRV1 to the agrobacterium solution containing the silencing expression vector is preferably 1:1. the agrobacterium of the present invention is preferably agrobacterium GV3101. The solvent of the agrobacterium liquid preferably comprises 10mmol/L MES, 20 mu mol/L acetosyringone, 10mmol/LMgCl ₂ and the balance of water. The OD value of the agrobacteria liquid containing TRV1 is preferably 0.8-1.0; the OD value of the agrobacterium tumefaciens bacteria solution containing the silencing expression vector is preferably 0.8-1.0.

The invention also provides a method for reducing the high temperature stress resistance of cotton, which comprises the following steps: the VIGS silencing system described in the above technical scheme is used to infect cotton plants. In the present invention, the means of infestation preferably comprises leaf injection. The invention preferably utilizes the VIGS silencing system to infect the true leaves of cotton plants; the area of infestation is preferably greater than 80% of the area of the true leaf.

The invention also provides a recombinant vector for improving the high temperature stress resistance of cotton, which comprises a basic vector and a GH_D07G0997 gene inserted into the basic vector. In the present invention, the base vector is preferably pCAMBIA3301.

The invention constructs the over-expression vector of the GH_D07G0997 gene, and leads the over-expression vector into cotton plants, and the result shows that the plants over-expressing the GH_D07G0997 gene have better stress resistance than the contrast, can endure the high temperature environment of 40 ℃, and can be used for screening and creating high temperature resistant cotton germplasm.

For further explanation of the present invention, the application of the cotton high-tolerance Wen Jiyin gh_d07G0997 provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Screening of candidate genes related to high temperature resistance

1. Materials and methods

1.1 Test materials

The test material was from 273 different cotton germplasm materials in 18 geographical areas, where: 139 parts of Xinjiang in China accounting for 50.92%; 17 parts of Henan, which accounts for 6.23 percent; 15 parts in total in the United states, accounting for 5.49%; 13 parts of Chinese and sub-five countries (Uzbexastan, kazakhstan, jier Ji Sisi, taginstan and Tukuman) total 4.76%; 12 parts of Shandong China accounting for 4.40 percent; 11 parts of Jiangsu in China accounting for 4.03 percent; 11 parts of Chinese Liaoning accounting for 4.03 percent; 10 parts of Hebei in China accounting for 3.66 percent; 8 parts of Shanxi of China accounting for 2.93 percent; 8 parts of Chinese Shanxi accounting for 2.93 percent; 8 parts of Hubei in China accounting for 2.93 percent; 5 parts of Gansu in China accounting for 1.83 percent; 5 parts of Sichuan China accounting for 1.83 percent; 3 parts of Hunan China accounting for 1.10 percent; 2 parts of Chinese Anhui accounting for 0.73 percent; 2 parts of Jiangxi China accounting for 0.73 percent; australian 2 parts, accounting for 0.73%; africa 2 parts, accounting for 0.73%. The germplasm has no direct relationship, and accords with the basic requirement of association analysis that the germplasm should be an unstructured population and have no direct relationship with each other. The test germplasm is provided by the upland cotton breeding team of the institute of economic crops of Xinjiang academy of agricultural sciences.

1.2. Test method and property investigation

The test was performed in 2017-2019 at a cotton comprehensive test station of Xinjiang agricultural academy of sciences of sixteen groups of first engineers in the city of Alar in Xinjiang for 3 consecutive years. A mechanical cotton picking film lower drip irrigation one-film six-row (10+66+10+66+10) cm planting mode is adopted, the theoretical plant number is 15500 plants/mu, the random block arrangement is carried out, the repetition is carried out for 3 times, the line length is 4.5m, and the field management is the same as that of a field.

The occurrence conditions of the 7-month and 8-month high-temperature weather in the region of the test development period are respectively as follows: 2017, 7, 8 > 30 ℃ for 43d and 35 ℃ for 16d;2018, 7, 8 > 30 ℃ for 47d and 35 ℃ for 21d;2019, 7, 8 > 30 ℃ for 53d and 35 ℃ for 14d.

13 Important traits of Plant Height (PH), initial node height (HFNFH), initial node number (HFNFB), fruit branch number (EFB), fruit node number (FN), single plant node Number (NB), pollen activity (PV), leaf Area (LA), chlorophyll content (Chl), stem bud number (DBs), falling rate (FB 3), falling rate (CB 3) and falling rate (DR) are investigated, and the specific investigation method is as follows:

plant Height (PH): after topping is finished and cotton plant height does not grow any more, measuring plant height in continuous 5 plants in the middle ten days of 8 months;

initial section height (HFNFH): selecting continuous 5 strains to measure the initial node height in the ten days of 8 months;

number of start nodes (HFNFB): selecting continuous 5 strains to measure the initial node number in the ten days of 8 months;

number of fruit branches (EFB): selecting 5 continuous plants to measure the number of fruit branches before harvesting;

fruit node number (FN): the number of fruit nodes was measured by selecting 5 consecutive plants before harvesting.

Number of Node Bell (NB) per plant: the number of node bell was measured by selecting 5 strains in succession before harvesting.

Pollen Viability (PV): and after high temperature stress, measuring pollen activity in tetrad period. Flowers were sampled and immersed in a2, 3, 5-triphenyltetrazolium chloride (TTC) solution, then left standing at room temperature for 1h, staining was stopped by adding 2% sulfuric acid, and pollen was photographed under a microscope with pollen viability = normal stained pollen/total pollen x 100%.

Leaf Area (LA): leaf areas of top, middle and bottom leaves were measured with LA-S leaf area meter for 5 consecutive strains and the average was taken as final phenotype data.

Chlorophyll content (Chl): chlorophyll content was measured with SPAD-502 chlorophyll meter for 5 consecutive strains, 3 times, and the average value was used as final phenotype data.

Number of Dry Buds (DBs): after high temperature stress, the number of the buds is measured by continuously measuring 5 strains within 10-15 days.

Falling rate (FB 3) of three fruit branches: the falling number of the three fruit branches is measured by 5 plants before harvest.

The number of the bell of the inverted three fruit branches (CB 3): the number of bell of three fruit branches was measured 5 plants before harvest.

Shedding rate (DR): ratio of empty fruit segments to fruit segment number.

A correlation map of phenotypic variation was generated using the ggplot package in R.

SLAF library construction and sequencing

Sampling was completed in 2021, and DNA extraction, library construction and sequencing of the sequenced samples were completed by beijing baimaike biotechnology limited.

1.4 Data analysis

After filtering low quality data and linker data from SLAF-seq sequencing data, the data is aligned to the cotton genome (https:// www.cottongen.org/data/download/genome_ tetraploid/TM-1) using BWA software, an index Bwaindex-aisref.fa is first constructed for the reference genome, then aligned using Bwamem-M method, sam is converted to Bam file using SortSam.jar in Picard tool set and ordered according to coordinates, and the number of SLAF tags and polymorphic SLAF tags on different chromosomes are counted. SNPs were developed using both the galkv 3.8 and Samtools methods, the SNP marker intersections obtained using both methods were used as the final reliable SNP marker dataset, and mutation annotation was performed using SnpEff to determine the location and effect of the mutation on the reference genome (intergenic region, genetic region or CDS region; synonymous mutation, non-synonymous mutation, etc.). To obtain a final set of high quality SNPs, a threshold minimum Minor Allele Frequency (MAF) of 0.05 and a maximum deletion percentage of 0.3 was set, and SNPs were filtered to yield a total of 81612 high quality SNPs for further population genetic analysis. And constructing a phylogenetic TREE by adopting IQ-TREE v1.6.12, and determining an optimal model as PMB+F+R7 according to the lowest BIC value ModelFinder. The population structure was calculated using FastSTRUCTURE v 1.0.0. To obtain loci associated with associated traits, a threshold minimum Minor Allele Frequency (MAF) of 0.05 and a maximum percent loss of 0.2 was set, and SNP was filtered to yield a total of 72662 high quality SNPs. Correlation analysis is performed by using CMLM in Tassel software packages, fast-LMM, EMMAX and other models. Setting Pvalues to be less than or equal to 0.1 and Pvalues to be less than or equal to 0.01 as thresholds and performing Bonferroni correction, and if no correlation site is obtained on the two threshold lines, adjusting the thresholds to be Pvalues to be less than or equal to 1E-0. And obtaining a remarkably-correlated SNP locus, and taking 100Kb upstream and downstream of the locus as a candidate region for subsequent candidate gene analysis. Candidate genes search annotation information through https:// cottonfgd.net/website, and search expression profile data of genes through https:// cotton.zju.edu.cn/website.

2. Results and analysis

2.1 Phenotypic trait analysis of different high temperature resistant terrestrial cotton seed resources

2.1.1.1 Descriptive statistical analysis of different high temperature resistant upland cotton germplasm phenotype traits

Descriptive statistical analysis results of 273 upland cotton variety resource group field investigation characters in 2017-2019 show that each investigation phenotype character accords with normal distribution. The variation coefficient of each investigation character in different years in the group ranges from 8.23% to 33.24%, which shows that each character in the group has rich genetic diversity and has certain research potential and value. The variation amplitude of the characteristics such as the activity of cotton pollen, the number of cotton bolls, the shedding rate, the number of fallen three fruit branches and the like at high temperature is larger, and the data discrete degree is probably increased due to the different high temperature resistance of group materials.

2.1.2. Analysis of phenotype character correlation of different high temperature resistant terrestrial cotton seed resources

Through correlation analysis on the average value of the 3-year-table-type characters, the distribution of the scatter diagram of the measured high-temperature-resistance-related agronomic characters is uniform, and the investigation character data is shown to have good representativeness. The plant height is obviously related to the initial node height, the number of fruit branches and the leaf area, and the related coefficients are respectively 0.374, 0.331 and 0.264. The plant height of the high-temperature-resistant material is obviously positively correlated with the initial node height, is obviously positively correlated with the number of fruit branches, and the plant height of the temperature-sensitive material is obviously negatively correlated with the leaf area.

High temperature resistance evaluation of 2.1.3.273 parts of upland cotton germplasm

Through comprehensive comparison of data average values in 3 years, 23 parts of varieties with better high temperature resistance are finally identified from 273 parts of germplasm materials, 113 parts of medium-resistance materials and 137 parts of temperature-sensitive materials, and the percentages are 8.42%, 41.39% and 50.18% respectively. The 23 parts of the identified high temperature resistant materials include new upland early No. 1, new upland early No. 34, new upland early No. 36, new upland early No. 48, new upland middle No. 14, new upland middle No. 32, new upland middle No. 47, new upland middle No. 64, new upland middle No. 68, C4744, farm land reclamation No. 5, source cotton No. 11, dai 80, middle cotton house 50, middle cotton house 2108, middle cotton 372, ji cotton 958, liao cotton No. 16, lu cotton No. 1, lu cotton No. 28, lu Yan cotton No. 29, yu cotton No. 19 and ai cotton. Wherein, the total of the Xinjiang self-bred variety and the Xinjiang historical variety is 14 parts, and the ratio is 60.87 percent.

2.2SLAF raw data statistics

A2161.76 Mb SLAF-seq sequence was obtained by SLAF-seq sequencing. By bioinformatic analysis 2133341 SLAF tags were obtained, of which polymorphic SLAF tags 532949, including 1313331 SNPs.

2.3 Structural analysis of different cotton germplasm populations

And constructing a phylogenetic tree by using a maximum likelihood method, dividing all materials into 6 branches, and finding that the optimal population structure of the population is 7 by combining the analysis result of the population structure, wherein the materials are from 7 different ancestor populations. The materials of Xinjiang's self-breeding are mostly clustered together, suggesting that the genetic basis of Xinjiang germplasm materials is narrower, and at the same time, the core germplasm has a more complex group structure.

2.4GWAS locating 7 personality-related QTLs

Carrying out whole genome association analysis on 7 high temperature resistant related phenotype characters such as chlorophyll content, leaf area, pollen activity, dry bell number, falling off number of three fruit branches, falling off rate and the like, and detecting 36 QTL loci containing 552 genes. Wherein the D subgenomic is relatively large, 21 loci are detected in total, and 336 genes are associated. The most detected D13 chromosome has total detected 4 QTL 97 genes, and the D09 chromosome has no detected QTL. The 15 loci 240 genes were detected in the a subgenomic, with the highest detection in a04, a07, each detecting 3 QTL loci, the associated numbers of genes being 72 and 12, respectively, whereas no corresponding QTL loci were detected in a01, a02, a03, a11 and a 12. The maximum number of genes related to chlorophyll is 196, and the pollen viability and the number of dry buds are related to 117 and 91 respectively, and the number of falling fruits, the falling rate and the leaf area are related to 53, 48, 38 and 33 genes respectively.

2.5GWAS locating 7 personality-related QTLs

For better screening candidate genes, the expression profile of the relevant genes was further analyzed using the cotton functional genome database (https:// cottonfg d.net /). Among the sites associated with 7 traits, 346 genes with higher expression in cotton stems, leaves, flower organs (stamens, calyx, flower receptacle) were found by expression profiling. Further according to the association result Manhattan diagram, the sites D08-5127676, D12-61662418 and D13-56652886 associated with leaf area, the site D1 ^2- 61662418 associated with pollen vitality, the sites D08-37941137, D12-42930672 and D13-4787908 associated with dry bud number, the site A06-21732616 associated with the bell formation of the three fruit branches and the sites A07-14871113 and D07-11614663 associated with the abscission number of the three fruit branches are respectively selected, and 108 genes are identified. It was found that the most chloroplast-related genes were functionally annotated, specifically as shown in Table 1 and FIGS. 1-2,

TABLE 1 candidate Gene annotation information Table

Example 2

Identification of high temperature resistance related candidate genes

1. Materials and methods

Through three years of identification of 273 parts of different high temperature resistant upland cotton germplasm resources, a high temperature resistant material 'Xinlandao No. 36' and a temperature sensitive material 'vehicle 61-72' are finally screened out as test materials.

2. Test method

After the test material seeds were sterilized in 15% hydrogen peroxide solution for 4 hours, they were rinsed 2 times with sterile water. The seeds were transferred to germination boxes (length: 12 cm. Times.width: 12 cm. Times.6 cm. High) containing sterilized sand, and 10 seeds were sown in each germination box at equal intervals, with a sowing depth of 2cm. After sowing, 300mL of deionized water was added to the box to saturate the sand with water, 200mL of water was added every 2 days, and the germination box was placed in an illuminated incubator (LRH 250-G, hangzhou instruments, inc.). The growth conditions of the seedlings were set as follows: photoperiod (16 h daytime, 8h nighttime); temperature (28 ℃ in the daytime, 20 ℃ at night); optical density (300. Mu. Mol m-2 s-1); relative humidity (75%). When cotton seedlings grow to the trefoil stage, high temperature stress at 40 ℃ is carried out for 0h, 4h, 8h and 12h in an illumination incubator, and each treatment is repeated for 3 times. Blade samples of 0h, 4h, 8h, and 12h after treatment were collected and labeled as T0, T4, T8, and T12 for New land early 36 material samples, and R0, R4, R8, and R12 for vehicle 61-72 samples, respectively. The sample was frozen in liquid nitrogen for 2h and transferred to a-80℃refrigerator for storage. The sample numbers are shown in Table 2.

Table 2 sample treatments and numbering

3. Transcriptome sequencing and data processing

3.1. RNA extraction and transcriptome sequencing of samples

Sample RNA extraction and transcriptome sequencing was undertaken by Beijing Baimichael Biotechnology Co.

3.2. Sequence alignment and differential gene analysis

Full length sequences were aligned with the reference genome using Minimap software to obtain non-redundant transcript sequences. The expression level of the gene was evaluated by comparing the number of the upper genes per 10000 reads. Differential gene screening is performed by using DESeq, fold Change is more than or equal to 2, and FDR is less than 0.01. The GO and KEGG functions of KEGG are enriched by adopting GOseq R software packages and KOBAS software respectively. The new coding gene was predicted using TransDecoder (v3.0.0) software.

Lncrna prediction

Because lncRNA does not encode proteins, four methods, CPC analysis, CNCI analysis, CPAT, PFAM protein domain analysis, were used to predict lncRNA for newly discovered transcripts, respectively. Transcripts selected from the four methods were further screened and a threshold (more than 200nt in length, more than two exons) was used to screen candidate lncRNA. Target gene prediction of lncRNAs was performed by two methods: (1) The expression of the neighboring genes of the lncRNA is regulated and controlled, and the expression is predicted mainly according to the position relation between the lncRNA and mRNA; (2) The lncRNA and mRNA are mainly used for target gene prediction by lncTar target gene prediction tools. The overlapping target genes predicted by the two methods were separated.

3.4. Weighted Gene Coexpression Network Analysis (WGCNA)

The expression values of DEGs screened above were used for WGCNA by R-package. The module is obtained by adopting an automatic network construction function module with default settings. And (5) analyzing the correlation between each module and the sample by using the Pearson correlation coefficient. The weighted network graph is further built by means of OmicShare tools (http:// www.omicshare.com/tools).

3.5. Real-time quantitative PCR (RT-qPCR)

To verify the accuracy of ONT transcriptome predicted gene expression, DEGs random 20 genes with expression levels higher than 10 were selected for RT-qPCR. Primers were designed using Primer6 (Primerbio software, usa). RT-qPCR was performed using a CFX96 fluorescent quantitative PCR system (Bio-Rad, USA) and FastKing one-step RT-qPCR (SYBR) kit (Tiangen, china). The internal reference gene is small nuclear subunit (SSU) rRNA, calculated by ²-^ΔΔCt method. Each sample 3 was repeated and the results were expressed as mean ± standard error variance after one-way analysis of variance, with significance set at P < value 0.05.

4. Results and analysis

4.1. High temperature stress treatment physiological index difference comparison analysis

4.1.1. ROS accumulation analysis of high temperature treated samples

Carrying out high-temperature stress treatment at 40 ℃ on the three-leaf-stage sample, wherein the temperature-resistant material has smaller wilting degree than the temperature-sensitive material from the aspect of the phenotype of the treated leaf; NBT staining of the high temperature stressed 12h inverted two-leaf sample revealed that the blue spot polymer product of ROS (O ^2-) accumulated on both New land early 36 and vehicle 61-72 leaves, and that vehicle 61-72 had more blue spot polymer than New land early 36. The area of the blue spot polymerization product of the vehicle 61-72 under high temperature stress for 12 hours is increased, the chromaticity is deepened, and the blue spot polymerization product is mainly distributed on mesophyll cells, veins and other parts, which is obviously more than that of Xinlunzao No. 36. The possible cause is breakdown of the defensive system in the body, abnormal ROS (O ^2-) function, resulting in excessive ROS (O ^2-) accumulation. And the accumulation of ROS (O ^2-) in leaf form of New land early 36 was less than that in vehicle 61-72, indicating that the defense mechanism against high temperature stress of New land early 36 was superior to that of vehicle 61-72 (FIG. 5).

The measurement of MDA, O ^2- and H ₂O₂ content in different treatment blades shows that the MDA and H ₂O₂ levels of different treatment sample vehicles 61-72 are higher than those of Xinlunzao No. 36. The MDA content of the two materials is reduced by high-temperature stress, the MDA content of the vehicle 61-72 is extremely obviously different from that of the new land No. 36 when the vehicle is stressed for 4 hours, the MDA content of the vehicle 61-72 is extremely obviously different from that of the new land No. 36 when the vehicle is stressed for 8 hours, and the MDA content of the vehicle 61-72 is increased when the vehicle is stressed for 12 hours, but no obvious difference exists between the two varieties. The H ₂O₂ content of the two varieties subjected to stress for 4 hours is the lowest, and the varieties are extremely obviously different. The two varieties are obviously different when the stress is 8H and the stress is 12H, and the content of H ₂O₂ is slightly lower than the stress is 8H. The O ^2- content was lower than New land early No. 36 for 0h treatment, but there was no significant difference, whereas for 4h treatment, 8h treatment, 12h the O ^2- content was higher for Yu Xinliu early No. 36 for 61-72, and the O ^2- content was decreased with increasing treatment time, and the two varieties reached very significant levels (FIG. 4)

4.1.2. Chlorophyll fluorescence quantitative analysis of high-temperature treated sample

By comparing the fluorescence parameters such as PS II initial fluorescence (Fo), PS II maximum photochemical efficiency (Fv/Fm), non-photochemical quenching coefficient (NPQ) and the like of the refractory material Xinlun Zao 36 and the refractory material vehicle 61-72 under the dark adaptation condition of the blade under the high temperature stress treatment of different time periods, the fact that the Fo of the vehicle 61-72 continuously rises along with the prolongation of the stress time and obviously rises after the stress is 8H in the high temperature stress treatment process is found, the fact that the Xinlun Zao 36 has no obvious rising trend indicates that the damage degree of the photosynthesis mechanism of the Xinlun Zao 36 is smaller than that of the vehicle 61-72. In the Fv/Fm index, after high-temperature stress treatment, both materials show a descending trend, and the descending amplitude of the temperature-sensitive material vehicles 61-72 is obviously larger than that of the temperature-resistant material Xinlunzao No. 36. In the NPQ index, both materials showed a continuous rise after high temperature stress treatment, and refractory material new land No. 36 was consistently significantly higher than vehicles 61-72 (fig. 5).

4.2. Transcriptome sequencing of different cotton varieties

Transcriptome sequencing gives a total of CLEAN DATA of about 187.53GB, up to 6.36GB per sample CLEAN DATA. Reads with linkers at both ends were judged to be full length sequences, the sequencing read length of each sample was greater than 884.33KB, and the average reads N50 length was 1024.17. Wherein the transcripts aligned to the reference genome are between 76.77% and 81.39%. Further clustering reads mapped to the reference genome and producing 120605 non-redundant sequences in total;

non-redundant sequences were annotated from 8 databases, generating 78601 genes. The annotation results showed that 55690, 26860 and 78569 genes were annotated from the GO, KEGG and Nr databases, respectively. Analysis of Nr homologous species distribution showed that the gene related species and the ratio of the genes annotated in this study were: upland cotton (51.22%), raymond cotton (17.87%) and island cotton (16.21%). GO enrichment of annotated genes was found to be significantly enriched in nuclear, cellular fraction and cell membrane composition, binding and catalytic activity in molecular function, metabolic processes in biological processes and cellular processes. The repeatability of the samples is evaluated by calculating the pearson correlation coefficient among the samples according to the expression level, and the samples have high intra-group correlation (0.91-1.00), which indicates that the samples have good biological repeatability. Principal component analysis showed that samples from different treatment groups exhibited discrete patterns, with the discrete patterns of tolerant samples being more pronounced, indicating that high temperature treatment had a significant effect on both cotton-type transcriptomes.

4.3 Identification of novel coding genes

The expression profile of the new gene showed different expression patterns at different times after high temperature treatment (fig. 6).

4.4 Identification and enrichment analysis of differential Gene

A total of 14 combinations were established to screen for differential genes, the groupings set in the R samples were: r0 vs R4, R0 vs R8, R0 vs R12, R4 vs R8, R8 vs R12; the groupings set in the T samples were: t0vs T4, T0vs T8, T0vsT, T4 vs T8, T8 vs T12; the analysis of the differences between R and T samples was grouped as: r0 vs T0, R4 vs T4, R8 vs T8 and R12 vs T12. 19600 genes were screened out of 14 combinations and the results are shown in Table 4.

TABLE 4 DEG statistics for each control group

By differential gene expression analysis, there are different expression patterns in different samples. The GO database annotated 14006 differential genes, GO enrichment analysis showed that the largest subclasses among the cell component plates were membrane, cell fraction, membrane fraction and organelle, containing 4986 genes (35.60%), 6103 genes (43.57%), 6009 genes (42.90%), 4346 genes (31.03%) and 4562 genes (32.57%), respectively. The molecular functional plate is mainly enriched in binding activity and catalytic activity, namely 6552 (46.78%) and 6492 (46.35%) genes respectively. In the biological process module, metabolic, cellular and single organism processes were enriched, accounting for 6559 (46.83%), 5978 (42.68%) and 3948 (24.97%) genes, respectively.

4283 Differential genes were co-annotated in the KEGG database, enriched in ribosomes (KO 03010), carbon metabolism (KO 01200), phytohormone signaling (KO 04075), amino acid biosynthesis (KO 01230) and endoplasmic reticulum protein processing (KO 04116) pathways.

To obtain a global map of the connections between these KEGG paths, a network between adjacent paths is built. The ribosomal pathway was found to contain the most diverse genes, which are involved in eukaryotic RNA transport and ribosomal biogenesis; protein processing in the endoplasmic reticulum pathway is associated with the proteasome pathway; carbon fixation in photosynthesis, the pathway of photosynthesis and its adjacent pathways (e.g., metabolic pathways and photosynthesis-antenna proteins) constitute a network. The phytohormone signaling pathway is associated with the most numerous neighboring pathways, such as tryptophan metabolism, diterpene biosynthesis and ubiquitin-mediated proteolysis.

In view of the fact that ribosomes are the most abundant pathway for differential genes, differential genes involved in this pathway were further analyzed. The results indicate that 526 differential genes belong to the ribosomal protein family and can be divided into 60S, 40S and 50S clusters. Among the ribosomal protein genes, the 60S ribosomal protein subfamily contains 298 differential genes, which are annotated to 45 members of the 60S ribosomal protein subfamily, such as 60S ribosomal proteins L40, L9, L18, and L26. The 40S ribosomal protein subfamily contains 196 differential genes. They are annotated into 34 members of the 40S small unit ribosomal protein subfamily, including 40S ribosomal proteins S10, S16, S18, and S30. The remaining 32 differential genes were annotated into 19 members of the 50S ribosomal protein, such as 50S ribosomal proteins L13, L9, and L34. It was found that they significantly respond to high temperature stress, the expression levels of these differential genes of temperature-sensitive varieties decreased with the increase of treatment time, and the expression levels of R12 were significantly lower than R0. However, the expression level of ribosomal proteins in T4 was significantly lower than T0, but was significantly up-regulated with prolonged high temperature treatment time.

4.5 Identification and enrichment analysis of differential Gene between varieties

To better investigate the different physiological responses of different varieties after stress, 4 combinatorial differential genes were analyzed, and from 4 combinations (R0 vs T0, R4 vs T4, R8 vs T8 and R12 vs T12) 5075 small unit ribosomal protein subfamilies were selected, of which the KEGG database annotated 1093 small unit ribosomal protein subfamilies, for which results enrichment analysis was similar to the KEGG enrichment results for the entire 40S small unit ribosomal protein subfamilies, which 1093 small unit ribosomal protein subfamilies were mainly enriched in ribosomes, carbon metabolism, phytohormone signaling and endoplasmic reticulum protein processing pathways. Analysis of the differential genes enriched in both phytohormone signal transduction and endoplasmic reticulum processing has led to the involvement of 53 40S small unit ribosomal protein subfamilies in endoplasmic reticulum pathway protein processing, 9 noted as transporter Sec61, 22 noted as 17 members of HSP, such as HSP17.5, HSP70. The results of the expression analysis show that 12 HSPs (such as HSP17.5, HSP70-17 and HSP 83) exhibit the same expression pattern, the expression level of R4, R8 and R12 is increased compared with that of R0, and in a T sample, the expression level of T4 is not significantly changed, and the expression level of the T8 and T12 samples is significantly increased compared with that of a T0 sample. These results indicate that there is a differential response in HSP between the temperature-sensitive variety and the temperature-resistant variety, and that there is a difference in the expression levels of Sec61 subsuit beta and gamma, with an increase in the expression levels of T8 and T12, and a decrease in the expression level of R12.

The 40S small unit ribosomal protein subfamily, which is enriched in the phytohormone signaling pathway, is primarily involved in auxin and ethylene responses and can be divided into 3 clusters depending on its expression pattern. In the first cluster, the expression level of ethylene response transcription factor 1B in T4 was significantly higher than in the other groups. Genes in the second cluster, such as EIN3-binding F-box protein 1-like and ethylene receptor-like, were expressed at the highest level in R12. The third cluster contains 6 auxin response protein genes (e.g., auxin-inducible protein and auxin response factor) and expression is significantly enhanced in T8 and T12.

4.6 Identification and enrichment analysis of differential Gene within variety

To investigate the differential gene between temperature-sensitive and temperature-resistant varieties, the differential gene analysis in the varieties was performed. 1194 common differential genes exist among the temperature sensitive materials R0 vs R4, R0 vs R8 and R0 vs R12, and 2333 common differential genes exist among the high-resistant Wen Pinchong T0 vs T4, T0 vs T8 and T0 vs T12.

KEGG and GO enrichment analysis was performed on the common differential genes, which were both enriched in ribosomes, protein processing in the endoplasmic reticulum and in the phytohormone signaling pathway. The GO result shows a differential enrichment path, the temperature-sensitive variety is mainly enriched in nucleosomes, protein folding and nucleosome DNA combination, the high-temperature resistant material is enriched in chloroplast thylakoid membranes, structural components and translation of ribosomes, and compared with the temperature-sensitive material, the paths of photosynthesis, light capture in an optical system I and the like are remarkably enriched in the high-temperature-resistant variety. Differential genes involved in these photosynthesis were screened and found to be mainly chlorophyll a-b binding proteins such as CAP10A, CAB and LHCB5. The genes have obvious expression difference between two varieties, and the expression quantity in T4 is obviously increased.

4.7LncRNA and target Gene prediction

By adopting four methods CNCI, CPC, pfam and CPAT, 5118 lncRNAs are identified, and the positions of the lncRNAs are divided into four types of lncRNAs positioned in intergenic regions, antisense, introns and coding regions, wherein the lncRNAs in the intergenic regions are in a main form and account for 87.1 percent. Analysis of the expression pattern of lncRNA most lncRNA showed higher expression patterns at 8h and 12h compared to 0h and 4h of high temperature treatment, which may be associated with heat stress. Target genes of lncRNA are predicted and analyzed to obtain 24462 target genes in total. Wherein 17087 and 4337 are annotated from the GO and KEGG databases, respectively. GO term statistics of the gene of interest show that metabolic processes, cellular processes, membranes, cells, binding and catalytic activity are most abundant.

KEGG enrichment analysis of the target gene showed significant enrichment of the pathways for ribosomes, phytohormone signaling, carbon metabolism, and endoplasmic reticulum protein processing, consistent with the results of DEGs analysis. Several representative target genes and their corresponding lncRNA were screened to show their relationship. As shown in FIG. 7, the relationship between 7 sets of lncRNAs and target genes is enumerated, for example, 60S ribosomal protein L24 is the target gene for LNCRNAS GH _A05G2717 and GH_A01G2277, and HSP90-1 and HSP80 are the target genes for lncRNAGH _A03G0301.

4.8WGCNA analysis

To study the gene regulation network of cotton under high temperature stress, WGCNA analyses were performed by screening 1240 differential genes in KEGG pathway (ribosomes, phytohormone signaling and endoplasmic reticulum protein processing), and the results indicated that 8 treatments could be clustered into 5 modules. Analysis of the module-trait relationship showed that the R12 and T4 groups were significantly associated with MEblue modules (r=0.94, p < 0.05) and MEyellow modules (r=0.84, p < 0.05), respectively.

Two weighted networks were plotted according to the weights between genes in the MEbule and MEyellow modules to find potential key genes. In these network diagrams, the larger the size of the nodes, the higher the connectivity of the genes. And 6 genes with highest connectivity are selected from MEbule modules to be used as hub genes. Of these 6 hub genes, gh_a13g1517.gene, gh_a04g0007.gene, and gh_d05g4056.gene are photosystem I reaction center subunit V (PSAG); GH_D07G1195.Gene is chloroplast photosystem I subunit O (PSAO); GH_A10G0169.gene and GH_D05G1713.gene are chloroplast oxygen evolution enhancer protein 3 (PSBQ). The MEyellow module generated 11 hub genes, namely 1 ribosomal protein (50S ribosomal protein L1, RPL 1), 3 heat shock proteins (HSP 70, HSP17.6 and HSP 83), 2 auxin signaling genes (IAA 1 and AUX 22D), 2 ATP synthase related genes (ATPC), 1 abscisic acid insensitive protein (ABF 1), 1 protein phosphatase (AHG 1) and 1 derlin 2.2.2 (DER 2.2). The analysis of the expression pattern of the hub genes showed that the expression levels of PSAG, PSBQ and PSAO in T0 and T4 were higher than those of R0 and R4, while the expression levels of the other hub genes were highest in R12.

4.9ONT transcriptome data RT-qPCR validation

And randomly selecting 20 genes to perform correlation analysis of ONT transcriptome data and RT-qPCR results so as to determine the reliability of the transcriptome data. The results showed that the ONT transcriptome and RT-qPCR data were significantly correlated with R ² = 0.6451 (p < 0.01), indicating that the ONT transcriptome data was authentic in this study.

Example 3

Candidate gene biological function verification

1. Materials and methods

1.1. Experimental materials

1.1.1. Experimental materials

PTRV1 (auxiliary vector) and pTRV2 (construction vector) used in the experiment, pTRV-CLA1 (positive control, coding 1-deoxyxylulose 5-phosphate synthase, participating in chloroplast development process, CLA1 gene mutant CLA1-1 having obvious albino phenotype) and pCAMBIA3301 vector are all stored in Xinjiang academy of agricultural sciences institute of economic crops laboratory. Agrobacterium GV3101 competent, E.coli DH 5. Alpha. Competent, LB medium, kanamycin, rifampicin, MS, mgCl2, acetosyringone purchased from Beijing Soy Corp; bamHI and SacI enzymes were purchased from Sieimer; YEB medium was purchased from hebo company; plasmid extraction kit, product purification kit, homologous recombination kit were purchased from the company Nanjinouzan; RNA extraction kit and reverse transcription kit were purchased from Beijing Baimeike Biotechnology Co.

1.1.2. Experimental instrument

Experimental instrument: gel imager, PCR instrument, metal bath, shaking table, ice maker, incubator, centrifuge, superclean bench, oven.

1.2. Experimental method

1.2.1. Experimental material planting

Cotton planting methods are referenced Li Xiuqing (Li Xiuqing. Cloning and functional identification of three genes related to resistance to verticillium wilt in cotton [ D ]. Xinjiang university of agriculture, 2019.).

The planting method of the arabidopsis is as follows:

(1) Taking part of Arabidopsis seeds, placing the Arabidopsis seeds into a 1.5mL sterile centrifuge tube, flushing with 70% alcohol for 3 times, 30s each time, and then using 2% sodium hypochlorite solution for the same disinfection treatment;

(2) Rinsing the arabidopsis seeds in the sterile centrifuge tube for 30s each time by using sterile water for 3 times;

(3) Uniformly placing the treated arabidopsis seeds in a 1/2MS solid culture medium, and air-drying the surface moisture by using sterile air of an ultra-clean workbench;

(4) Sealing the culture dish by using a sealing film, and placing the culture dish in a refrigerator at the temperature of minus 4 ℃ for vernalization;

(5) After vernalizing for 3d, placing the mixture in a culture box at 25 ℃ for culture until two leaves grow well;

(6) Transplanting the Arabidopsis plants with complete roots into a flowerpot for culture under the condition of 16h light/8 h dark at 25 ℃.

1.2.2. Silencing fragment PCR amplification

Specific fragments of GH_D07G0997 (BamHI and SacI) genes are inserted into the silencing vector pTRV2 enzyme cutting sites, specific enzyme cutting primers are respectively designed at two ends of the specific fragments, protective bases are added at two ends of the enzyme cutting primers, and PCR amplification is carried out, wherein the information of the primers is shown in Table 5.

The PCR reaction procedure was as follows: 3min at 95 ℃;95 ℃ 30s,58 ℃ 30s,72 ℃ 90s,35 cycles; 72 ℃ for 10min;

TABLE 5 primer information

1.2.3. Silencing vector construction

Plasmid DNA of GH_D07G0997 gene is used as a template, target gene fragments are amplified by PCR, and simultaneously escherichia coli bacterial liquid containing pTRV2 empty vector is cultured for plasmid extraction. The restriction endonuclease is utilized to construct a plant silencing vector pTRV2-GH_D07G0997 for the extracted pTRV2 vector plasmid, and the vector pTRV2-GH_D07G0997 is transformed into agrobacterium and stored at the temperature of minus 20 ℃ for standby. For specific steps reference Wang Yi (Wang Yi. Functional identification of GhMYB4, gbTCP and GbTCP in cotton drought and salt stress response [ D ]. University of agriculture, xinjiang, 2022.) the ligation system is: 8. Mu.L of linearized vector, 6. Mu.L of insert, 4. Mu.L of 5 XCE II Buffer, exnase II. Mu.L, and 20. Mu.L of ddH ₂ O.

Preparation of a ViGS-transformed bacterium solution

(1) Taking out the agrobacterium preserved at-20 ℃, sucking 50 mu L, placing the agrobacterium in 5mL of YEB liquid culture medium containing 50mg/mL kan+, rif+ antibiotics, and culturing at a constant temperature of 220rpm/min at 28 ℃ for 12-16 h.

(2) 1ML of bacterial liquid is taken out from the previous step and transferred into 50mL of YEB liquid culture medium containing 50mg/mL of kan+, rif+ antibiotics, 10mmol/LMES and 20 mu mol/L acetosyringone according to the proportion of 1:50, and the bacterial liquid is subjected to shaking culture at a constant temperature of 28 ℃ and 220rpm/min until the OD600 is between 0.8 and 1.0.

(3) Centrifuging the large-shaking bacterial liquid at 4000rpm/min for 12min, fully enriching bacterial precipitate, and discarding the supernatant.

(4) After enrichment of thalli, fully dissolving fresh thalli in a heavy suspension containing 10mmol/L MES, 20 mu mol/L acetosyringone and 10mmol/L MgCl2, adjusting to OD600 = 0.8-1.0, loading into a sterile centrifuge tube, packaging with tinfoil paper, standing for 3 hours at room temperature in a dark place, and then using for silencing injection.

Genetic transformation of VIGS

(1) The prepared bacterial liquid is placed in a sterile culture dish (TRV 1: TRV2\TRV 2-CLA=1:1) and fully and uniformly mixed.

(2) The bacterial liquid is injected into two real leaves of the receptor material (negative control 20 strains, positive control 20 strains and treatment group 20 strains) by using a sterile injector, and the injection area is ensured to be more than 80 percent.

(3) After the receptor material was injected, the normal culture was performed after dark culture for 24 hours. After the albino phenotype appears in the negative control group, a high temperature stress experiment at 40 ℃ is carried out, and the phenotype change is observed.

Preparation of pCAMBIA3301 transformed bacterial liquid

(1) Mu.L of pCAMBIA3301-GH_D07G0997 Agrobacterium solution stored at-20 ℃ is sucked into 1mL of YEB liquid culture medium which is added with 50mg/mL of kan+, rif+, gen+ antibiotics in advance, and the temperature is kept constant at 28 ℃ and 180rpm/min, and the mixture is cultured until the OD600 = 0.8.

(2) Adding the cultured bacterial liquid into a fresh liquid YEB culture medium according to the proportion of 1:100 for propagation, and culturing until the OD600 = 1.0-1.2.

(3) And centrifuging the bacterial liquid after propagation for 10-15 min by using a centrifuge at 4000rpm/min to obtain enriched bacterial precipitate.

(4) During infection, the enriched thalli precipitate is resuspended by using a suspension containing 10% and 5% of sucrose, 0.05% of MS and 0.02% of MgCl2 are added into the 5% of sucrose suspension, and the mixture is resuspended until OD600 = 0.8-1.0 and then transferred into a sterile triangular flask for standby (the day of infection is selected for the purpose of guaranteeing the activity of the bacteria).

1.2.7. Transformation of Arabidopsis thaliana

(1) The flowers which are possibly fertilized and the pods which are formed at the beginning are cut off by scissors in the flowering stage of the wild arabidopsis thaliana (WT) in the culture room. And (3) using heavy suspension containing pCAMBIA3301-GH_D07G0997 thalli, selecting arabidopsis thaliana with better growth vigor for infection, infecting arabidopsis thaliana flowers for 1-2 min, performing dark culture for 24h after infection, transferring to 25 ℃ and continuing culture in a 16h light/8 h dark environment until seeds are harvested (infecting for 1-2 times according to actual situation).

(2) After the T1 generation seeds are planted indoors, sampling is carried out when 4 true leaves grow out on the plants, PCR experiment detection is carried out, the transgenic positive plants which are detected preliminarily are reserved, and culture is continued until the T2 generation seeds are harvested.

(3) And (3) planting the T2 generation seeds indoors, and observing the phenotype of the T2 generation seeds by high-temperature treatment at 40 ℃.

Silencing vector for qRT-PCR detection

When the positive control plants showed albino phenotype after 10d injection, control and treatment groups of leaf RNAs were extracted, extracted and reverse transcribed, and silencing efficiency was detected by qRT-PCR.

2. Results and analysis

2.1. Screening of high temperature resistant candidate genes

By combining the candidate genes related to the GWAS with transcriptome data analysis, only GH_A06G0866, GH_D07G0997, GH_D07G0998 and GH_D12G1432 in the candidate genes related to chloroplasts are found to be differentially expressed, and the rest candidate genes are not differentially expressed or not expressed. Wherein GH_A06G0866 up-regulates expression only in the R0 vs R12 packet. GH_D07G0997 up-regulated expression in R0 vs R12, R0 vs R4, R0 vs R8, T0 vs T12 comparison group. GH_D07G0998 up-regulated expression in the comparison group T0 vs T4, T4 vs T12 and down-regulated expression in T4 vs T8. GH_D12G1432 down-regulates expression in R0 vs R12, T0 vs T8, T0 vs T12, T4 vs T8, up-regulates expression in R4 vs R12, R4 vs T4, T0 vs T4, T4 vs T12. 4 QTL loci and markers are shown in Table 6.

Table 6QTL locus and markers

Screening of the transcriptome expression level revealed that the factor of difference in the expression level of chlorophyll-related gh_d07G0997 in resistant and sensitive varieties was 1.5 or more (fig. 8).

The homology of the candidate gene in arabidopsis thaliana is shown to be AT4G13010, the similarity is 98%, the gene belongs to a redox enzyme zinc binding dehydrogenase family protein member, has the activity of redox enzyme, is combined with zinc ions to participate in the redox process, is positioned in chloroplast thylakoid membranes, chloroplasts, plasma membranes and vacuoles (Table 6), the gene structure is shown in figure 9, and the three-level structure prediction is shown in figure 10.

TRV-CLA (Positive control) phenotype

The phenotype was observed 10d after the injection at the time of flattening the two cotyledons, and the result shows that the leaves of the positive control plant are wilted and part of the leaves are white spots, even the leaves are whitened completely, meanwhile, the negative control plant grows better than the positive control plant, and the stem of the positive control plant is whitened to a certain extent (figure 11).

2.3. Silencing efficiency detection

After the positive control is whitened, taking the leaves of the negative control group, taking cotton leaves with the growth conditions consistent with those of the cotton leaves injected with the VIGS empty vector as a control, and detecting the silencing efficiency of the target gene by using a qRT-PCR experiment, wherein the detection result shows that the expression quantity of the target gene is obviously lower than that of the negative control group, and the target gene is successfully silenced (figure 12).

2.4. High temperature stress experiment at 40 ℃ for silencing plants

The cotton injected with the VIGS empty vector and the cotton injected with the target gene are subjected to high temperature stress at 40 ℃, and the fact that four plants have wilting at different degrees is found, the wilting degree of TRV:00 is not obvious, the wilting phenomenon of the TRV: GH_D07G0997 plant is obvious, and even the stem tip of the whole plant sags due to the wilting of the stem (figure 13).

2.5. Determination of physiological index of silenced GH_D07G0997 Gene plant

After 40 ℃ treatment, the gh_d07G0997 silenced plants were compared to negative control plants for peroxidase, leaf moisture, catalase, proline, superoxide dismutase, and malondialdehyde levels, and the results are shown in fig. 14.

As can be seen from fig. 14, the peroxidase content of the high temperature treated plants was higher than that of the untreated plants, but the empty load in each group was not significantly different from that of the plants containing the target gene, which might indicate that the experimental group can respond to the high temperature stress, and the plants might possibly remove the peroxy anions in the body by increasing the peroxidase content, so as to improve the high temperature tolerance of cotton; the results of leaf moisture content showed that the control and experimental groups in both the untreated and high temperature treated groups showed significant differences and that the moisture content of the silenced plants was significantly lower than the control, which also indicated that the gene might play a role in cotton response to high temperature; the results of catalase content show that the enzyme content of plants which are not subjected to high-temperature treatment is not obviously different, but the enzyme content of plants in untreated groups is obviously different, and the enzyme content of control plants in the groups is obviously higher than that of plants with silent genes; superoxide dismutase content shows that the enzyme content of plants which are not subjected to high temperature treatment is higher than that in the groups which are subjected to high temperature treatment, but the enzyme content in the treated groups and the untreated groups are not obviously different; the malondialdehyde results show that the malondialdehyde content in the untreated group is higher than that in the high-temperature treated experimental group, and that the malondialdehyde content of the plants in the control group which is not subjected to high-temperature treatment is obviously different, and the enzyme content of the control group is obviously higher than that of the silent plants; there was no significant difference in proline content within each group in both the untreated and high temperature treated groups in the proline assay results.

2.6. Transgenic Arabidopsis phenotype identification

PCR amplification screening of positive plants was performed on transgenic plants using the primers of Table 5, and the results showed that the amplified bands were consistent with the target gene bands (FIG. 15);

After the plant over-expressing GH_D07G0997 is treated at 40 ℃, the plant over-expressing Arabidopsis thaliana grows better than the wild type plant, and the leaf of the wild type plant has obvious wilting phenomenon. (FIG. 16).

From the above, it can be seen that plants that silence the gh_d07G0997 gene have poor stress resistance compared to the control, and plants that overexpress the gh_d07G0997 gene have better stress resistance. GH_D07G0997 responds to high temperature stress by affecting plant water and malondialdehyde, does not participate in the proline biosynthesis pathway, and plays a positive regulation role in the high temperature stress process.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The application of the cotton with high-tolerance Wen Jiyin GH_D07G0997 comprises one or more of the following (a) - (c);

(b) Screening high temperature resistant cotton seeds;

(c) Creating high temperature resistant cotton germplasm;

the GH_D07G0997 has an accession number XP_040953438.1 at NCBI.

2. The use of claim 1, wherein creating a high temperature resistant cotton germplasm comprises: overexpressing the cotton high temperature resistant gene to obtain a high temperature resistant cotton plant;

the cotton includes upland cotton;

The ambient temperature for the application was 40 ℃.

3. A silencing expression vector for reducing the high temperature stress resistance of cotton, which is characterized by comprising a gene silencing vector skeleton and a silencing gene fragment inserted into the gene silencing vector skeleton;

the silent gene fragment is a characteristic fragment of the GH_D07G0997 gene;

4. The silencing expression vector of claim 3, wherein the gene silencing vector backbone comprises pTRV2.

5. A VIGS silencing system for reducing the high temperature stress resistance of cotton, comprising an agrobacterium solution comprising TRV1 and an agrobacterium solution comprising the silencing expression vector of claim 3 or 4.

6. The VIGS silencing system of claim 5, wherein the volume ratio of the agrobacterium solution containing TRV1 to the agrobacterium solution containing the silencing expression vector is 1:1, a step of;

The OD value of the agrobacteria liquid containing TRV1 is 0.8-1.0;

7. A method for reducing the high temperature stress resistance of cotton, comprising the steps of: infection of cotton plants with the VIGS silencing system of claim 5 or 6.

8. The method of claim 7, wherein the means for infestation comprises leaf injection.

9. A recombinant vector for improving the high temperature stress resistance of cotton, which is characterized by comprising a basic vector and a GH_D07G0997 gene inserted into the basic vector.

10. A method for improving the high temperature stress resistance of cotton, comprising the steps of: introducing the recombinant vector of claim 9 into a cotton plant.