CN118813645A - A cotton Verticillium wilt resistance gene Gbar_4CL1 and its application - Google Patents

Abstract

The present invention relates to a cotton Verticillium wilt resistance gene Gbar_4CL1 and its application. The present invention performs map-based cloning on the gene of cotton Verticillium wilt resistant material M34, and identifies the cotton Verticillium wilt resistance gene Gbar_4CL1 for the first time. The gene has important value for theoretical research such as analyzing the molecular mechanism of cotton Verticillium wilt resistance. The Gbar_4CL1 gene can improve cotton's resistance to Verticillium wilt, and has important practical application significance for cotton Verticillium wilt resistance breeding.

Description

Cotton verticillium resistance gene Gbar_4CL1 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a cotton verticillium resistance gene Gbar_4CL1 and application thereof.

Background

Cotton is an important economic crop, not only provides an important natural fiber raw material for textile industry, but also has important application in the fields of national defense, medicine, automobile industry and the like, and has an irreplaceable position in national economic development. The discovery of verticillium in cotton was originally traced back to the united states at the beginning of the 19 th century, from which cotton producing areas around the world had profound damage over time.

Verticillium wilt is known as "cancer" of cotton and exists in major cotton producing areas around the world. The verticillium wilt can be infected in the whole growth period of cotton, and the main symptoms are that leaves lose green and turn yellow, wilt, withered and fall off, plants become short, bolls become small and the like, and even the cotton can die. The average yield reduction of cotton caused by verticillium is about 10-35%.

Verticillium wilt is a major disease faced by current cotton production and will severely reduce fiber yield and quality. The physicochemical control and biological control only can temporarily control the spread of verticillium wilt, reduce the extent of injury, and the cotton variety with high verticillium wilt resistance is the most fundamental and effective control measure at present. The separated population is utilized to carry out positioning research on verticillium wilt resistance related characters, and meanwhile, genes related to verticillium wilt resistance are identified, so that a certain theoretical guidance is provided for cultivation or improvement of high-resistance cotton varieties.

Disclosure of Invention

The invention provides a cotton verticillium wilt resistant material M34 and application of a disease resistance gene Gbar_4CL1 carried by the material in regulating and controlling cotton verticillium wilt resistance, and solves the problems of germplasm and gene deficiency encountered in cultivation of upland cotton varieties with verticillium wilt resistant characteristics in the prior art. The invention discovers a cotton verticillium wilt resistant material M34, identifies a cotton verticillium wilt resistance regulating gene Gbar_4CL1 carried by the cotton verticillium wilt resistant material M34 through a map-based cloning technology, further proves that the Gbar_4CL1 gene is expressed in high quantity after being induced by verticillium wilt, and can obviously reduce cotton verticillium wilt resistance in cotton by VIGS, so that the gene plays an important role in regulating cotton verticillium wilt resistance.

The invention provides a gene Gbar_4CL1 for regulating and controlling verticillium wilt resistance of cotton, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. The invention also provides a coding protein for regulating and controlling the cotton verticillium wilt resistance gene, wherein the CDS sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 3.

It will be appreciated that modifications to the Gbar_4CL1 gene sequence, without altering the amino acid sequence, are also within the scope of the invention, given the degeneracy of the gene expression control regions and codons.

The invention also provides a product comprising any one of the following a 1) to a 3):

a1 An expression cassette encoding the above gene; a2 A recombinant vector comprising the expression cassette of a 1); a3 A recombinant microorganism comprising the expression cassette of a 1) or the recombinant vector of a 2).

The invention also provides application of the gene or the product in enhancing cotton disease resistance, preparing products for enhancing cotton disease resistance or cultivating cotton disease resistance varieties. Further, the disease resistance is cotton verticillium wilt resistance, and cotton comprises upland cotton and island cotton.

The invention also provides a method for enhancing the disease resistance of cotton or cultivating cotton disease resistance varieties, and the genes or the products are introduced into cotton for expression. Further, the disease resistance is cotton verticillium wilt resistance, and cotton comprises upland cotton and island cotton.

The invention also provides a method for cultivating transgenic plants with reduced verticillium resistance, comprising the following steps: inhibiting the expression of Gbar_4CL1 gene shown in SEQ ID NO. 1 in the acceptor plant to obtain transgenic plant; the transgenic plant has lower resistance to verticillium wilt than the recipient plant.

Further, the transgenic plant has lower resistance to verticillium than the recipient plant is represented by b 1) or b 2) below: b1 The susceptibility of the transgenic plant is higher than that of the recipient plant; b2 A transgenic plant has a higher disease susceptibility index than the recipient plant.

Further, the expression of Gbar_4CL1 gene shown in SEQ ID NO:1 in the recipient plant is inhibited as follows: the TRV:00 vector of the Gbar_4CL1 gene is constructed by utilizing the VIGS technology, and the Gbar_4CL1 gene in a receptor plant is silenced. Further, the plant includes cotton, upland cotton, and island cotton.

The invention also provides an application of the Gbar_4CL1 gene and the encoded protein thereof in improving cotton verticillium resistance. The cotton verticillium wilt-resistant material M34 is inoculated with verticillium wilt bacteria V991, and the expression level of Gbar_4CL1 gene is increased, so that the cotton verticillium wilt-resistant material M34 is disease-resistant; silencing the expression of gbar_4cl1 by VIGS technology reduces cotton resistance to verticillium.

The beneficial effects are that: the invention performs map cloning on the gene of the cotton verticillium wilt resistant material M34, identifies the cotton verticillium wilt resistant gene Gbar_4CL1 for the first time, discovers that the gene has a difference between the material M34 and the material E22, and mainly shows a difference in promoter sequence, so that the gene expression amounts of the two materials have a difference, wherein the Gbar_4CL1 gene of the M34 has high expression so as to show verticillium wilt resistance. The gene has important value for theoretical researches such as molecular mechanism for analyzing cotton verticillium wilt resistance. The material and the gene can obviously improve the resistance of cotton to verticillium wilt, and have important practical application significance for cotton verticillium wilt-resistant breeding.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows that the island cotton introduction line M34 was superior to the upland cotton jaw cotton 22 (E22) in terms of verticillium resistance in greenhouse planting. A: e22 status on day 14 after V991 inoculation; b: status of M34 on day 14 after V991 inoculation; c: e22 and M34 disease refer to investigation and differential significance analysis; d: e22 and M34 epicotyl stem segments are recovered to culture; e: and E22 and M34 bacteria content detection.

FIG. 2 is a map-based clone of Gbar_4CL 1. A: preliminary positioning of Gbar_4CL 1; b: the island cotton introduction fragment in the island cotton introduction line M34; c: frequency histogram of disease resistance of F ₂ group; d and E: fine positioning of gbar_4cl1; f: differential gene wien diagrams of the same parent in the same period and different parents in the same period in a pairwise comparison mode; g: differential genome data expression levels present in candidate regions.

FIG. 3 shows the response of the Gbar_4CL1 gene to verticillium infection at E22 and M34. A: tissue expression pattern of gbar_4cl1 at E22 and M34; b: the expression quantity of Gbar_4CL1 is changed in different time periods after E22 and M34 are inoculated; c: gbar_4CL1 expression level was varied without inoculating V991 to E22 and M34.

FIG. 4 shows the sequence differences between the promoters and CDS regions of the Gbar_4CL1 gene at E22 and M34.

FIG. 5 is a graph showing the effect of silencing Gbar_4CL1 gene on cotton verticillium resistance. A: TRV the albino phenotype of CLA on day 14 of V991 infection; b: disease phenotype of TRV 00 and TRV Gbar_4CL1 at day 14 of V991 infection; c: the expression level of Gbar_4CL1 at TRV:00 and TRV: gbar_4CL 1; d: investigation of TRV 00 and TRV Gbar_4CL1 disease indexes; e: plant stem culture after 14 days of V991 infestation; f: detecting the bacterial content of plant stems after 14 days of V991 infection; g: browning of plant stem vascular bundles 14 days after V991 infestation.

FIG. 6 is a graph depicting silencing Gbar_4CL1 disrupting lignin deposition and reducing cotton verticillium resistance. A: paraffin sections of TRV 00, which are the same image from top to bottom, are enlarged by 40X, 100X and 200X; b: TRV Gbar_4CL1 paraffin section, from top to bottom is the same image, enlarged 40X, 100X, 200X cases.

Detailed Description

The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1 greenhouse disease resistance identification of E22 and M34 for V991

(1) Plant material sources and planting methods: the parent materials used in this experiment were upland cotton jaw cotton 22 (Emian, hereinafter E22) and laboratory pre-constructed island cotton chromosome segment substitution material M34. The parent materials E22 and M34 were grown in the agricultural university of China test field at 2022, 4 months, and obtained by self-crossing. And respectively selecting 200 large and full seeds E22 and M34, carrying out germination accelerating treatment, sowing in seedling boxes, uniformly sowing 30 seeds in each seedling box, selecting cotton seeds which are exposed and consistent in germination, sowing the seeds on demand, applying vermiculite, and covering a preservative film for preserving moisture and heating. After cotyledon flattening, removing the film, washing off vermiculite from cotton seedling, transferring to 1/2MS nutrient solution, transferring to artificial climatic chamber (28 deg.C, 16H illumination and 8H darkness) for culturing for 4-5 days, and performing V991 inoculation treatment.

(2) Activation and culture of verticillium: transferring the materials to 1/2MS nutrient solution, uniformly coating 100uL V991 spore liquid stored at the temperature of minus 70 ℃ of the subject group to PDA (potato dextrose agar) culture medium in an ultra-clean workbench, inversely culturing for 3-5 days in a constant temperature incubator at the temperature of 25 ℃ after complete air drying, inoculating a proper amount of mycelium blocks to a proper amount of Czapek culture medium, culturing for 3-5 days in a shaking table at the temperature of 25 ℃ and 180rpm, filtering the spore suspension by 2 layers of gauze, fully mixing uniformly, diluting by 10 times, adopting a 5-point sampling method, counting the number of spores by using a blood cell counting plate under an optical microscope, calculating the concentration of the spore suspension, diluting the concentration of the spore suspension to 5X 10 ⁵/mL by using distilled water, and uniformly mixing for verticillium inoculation.

(3) Inoculating verticillium: cotton seedlings with consistent growth vigor are selected, and the spore suspension root dipping method is adopted to inoculate verticillium wilt bacteria V991. Mock: immersing the root system of the treated seedling in 500mL distilled water for 2min; and (3) inoculation: cotton seedling roots were immersed in 500mL of spore suspension at a concentration of 5 x 10 ⁵ pieces/mL, during which time the spore suspension was gently shaken and root immersed for 2min.

The MOCK group and the seedling of the inoculation group are planted in nutrient soil (nutrient soil: vermiculite=1:1, v:v) and placed in an illumination culture room with a light cycle of 16H illumination and 8H darkness at 25 ℃. And counting the disease condition when the cotton seedlings are inoculated for 9-12 days and have verticillium wilt disease symptoms. The disease index of each treatment is counted through plant diseases such as leaf dehydration, wilting, withering, yellowing and falling off.

Disease Index (DI): statistical methods referring to "cotton verticillium wilt resistance identification protocol" (NY/T2952-2016), the degree of verticillium wilt occurrence of plants was classified into 5 disease classes: 0. stages 1, 2, 3, 4. The disease index calculation formula: disease Index (DI) =100×Σ (leaf number of each stage×representative value of each stage)/(total leaf number of investigation×representative value of highest stage).

(4) Recovery culture of verticillium: taking the stalks of the same part (hypocotyl/first section) of cotton seedlings about 16 days after inoculation, diluting 84 disinfectant (luwa, available chlorine content 8000 mg/L-10000 mg/L) with sterilized water in an equal volume for 1 time, immersing the stalks in the diluted disinfectant for 5min, slightly shaking the disinfectant during the period, rinsing the sterilized solution for 3-4 times with sterile distilled water to wash off redundant disinfectant on the surfaces of the stalks, placing the stalks on sterilized filter paper in the ultra-clean workbench, cutting the stalks into 2-3 mm-sized stalks by a sterile cutter after the stalks are dried, placing the cut stalks on a PDA culture medium according to material partition, inverting the culture medium in a culture box at 25 ℃ for 2-4 days, observing the growth condition of hyphae, and photographing.

(5) Verticillium bacteria-containing amount measurement: about 16 days after inoculation, taking one section of the inoculated cotton Miao Di, rapidly placing the inoculated cotton into liquid nitrogen, and preserving the inoculated cotton in a refrigerator at the temperature of minus 80 ℃ for later use. After the liquid nitrogen is fully ground, DNA is extracted by a DNA extraction kit (FastPure Plant DNA Isolation Mini Kit, vazyme, DC401-01, application method see product instruction book), the template is diluted to 10 ng/mu L to be used as the template after the concentration is measured, ghUBQ (Ghir _A11G 011460) is used as an internal reference gene, ITS1-F and ST-VE1-R are verticillium fungus specific primers, and a qRT-PCR method is used for detecting fungus biomass in the stems. Wherein ITS1-F: AAAGTTTTAATGGTTCGCTAAGA, ST-VE1-R: CTTGTGTCATTTAGAGGAAGTAA.

(6) Greenhouse verticillium resistance identification of E22 and M34: the E22 and the M34 are infected by the fallen leaf type V991, planted in a greenhouse, and observed after 10 days of inoculation, the results are shown in the figure 1A and the figure 1B, the disease conditions of the two are greatly different, the plants of the experimental group of the E22 are subjected to large-area yellowing, water loss and wilting, and the disease conditions of the M34 are lighter in comparison with the yellowing, water loss and wilting. The difference significance analysis is carried out by using the disease index obtained by using the t test, the result is shown in fig. 1C, and the result shows that the disease index of E22 reaches nearly 70%, the disease index of M34 is in sharp contrast with the disease index, the disease index is less than 40%, and the disease index of the two disease indexes show significant difference. After plant disease resistance observation and investigation of disease finger ends, the epicotyl of cotton was excised for recovery, and the results are shown in FIG. 1D, which shows that many stem segments of E22 have regrown to V991, while M34 has many normal stem segments. The hypocotyl of the infected plant was excised, DNA was extracted, the bacterial load was detected by qRT-PCR, and the difference significance analysis was performed by t-test, the results are shown in fig. 1E. As can be seen from FIG. 1E, the bacteria content of E22 and M34 are significantly different, and the bacteria content of E22 is about 0.75 and the bacteria content of M34 is about 0.35. Through data analysis and phenotypic investigation, E22 disease resistance is shown to be significantly lower than M34.

Example 2gbar_4cl1 localization and mapping cloning

(1) Initial positioning of gbar_4cl1: the method comprises the steps of taking tetraploid high verticillium resistant island cotton seed 3-79 as a donor parent, taking a verticillium sensitive upland cotton variety E22 as an acceptor parent, firstly taking E22 as a female parent and taking island cotton 3-79 to obtain F ₁ generation, then taking E22 as a recurrent male parent to carry out continuous backcross for 6 generation, and carrying out selfing for 1 generation, thus finally obtaining 3-79 introgression line populations comprising 148 multi-segment introduced lines and 177 single-segment introduced lines. The introgression fragments of these 325 3-79 introgression lines were identified using genome resequencing, and a total of 2 3-79 chromosome-introduced fragments were identified, located on the A05 and D12 chromosomes, respectively (FIG. 2B). And (5) identifying and screening the disease-resistant introduction line M34 in the field of the introduction line.

The introduction line M34 (disease resistance grade is 1 grade) with the introduction fragment of the corresponding sea island cotton 3-79 and recurrent parent E22 number (disease resistance grade is 3 grade) are hybridized and selfed to obtain F ₂ generation separation groups (1964 single plants), and the disease resistance grade of each single plant is investigated in the mature period. The results showed that the disease resistance levels of most individual plants in the F ₂ population lie between level 1 and level 2 and exhibit a biased peak distribution (fig. 2C), with 310 level 0 resistant plants, 690 level 1 resistant plants, 407 level 2 resistant plants, 297 level 3 resistant plants, 260 level 4 resistant plants. Indicating that the verticillium wilt resistance of M34 has main gene control.

InDel markers are designed and developed by introducing fragments on A05 and D12 chromosomes through early resequencing data, 300 pairs of InDel markers are designed and developed on the A05 chromosome together, one molecular marker with the average distance of 0.1Mb is selected by utilizing parents, the available polymorphic marker 40 pairs are screened out, through continuous acquisition of genotype data and combination of F ₂ phenotype data, the situation that chromosomes are crossed and interchanged on upper and lower markers of K175 and K268 is found, and high-density marker development is carried out at the position. 50 pairs of InDel markers are developed on the D12 chromosome, but InDel markers with linkage effects on the population cannot be developed, and finally the chromosome where the gene is located is focused on A05. Through development of InDel molecular markers and acquisition of genotypes, and by combining phenotype investigation data of F ₂ groups, the QTL is obtained to exist in an A05 chromosome (figure 2A), and the physical position reaches about 11 between markers K183 and K208 at (K183_F:5'AGATACAAAGAGTAAGGGATAGGG 3'、K183_R:5'TGACTTGAATGATAGTAGTTAAAGTTCT 3';K208_F:5'AAAAATTGACGTGGTT GATAGAATA 3'、K208_R:5'TTTTTGTAGTGGTTCATGTAGGTTAA 3'),LOD.

The interval is finally reduced from the original 6.2Mb interval to 129kb in size. There are 11 candidate genes in this region, ：Gbar_A05G000520、Gbar_A05G000530、Gbar_A05G000540、Gbar_A05G000550、Gbar_A05G000560、Gbar_A05G000570、Gbar_A05G000580、Gbar_A05G000590、Gbar_A05G000600、Gbar_A05G000610、Gbar_A05G000620、Gbar_A05G000630、Gbar_A05G000640、Gbar_A05G000650、 and gbar_a05g000660, respectively. Among these are many genes involved in plant stress resistance, such as gbar_a05g000520 (gbar_syn61) involved in osmotic stress tolerance and regulation of the stomatal response by abscisic acid (ABA), and together with gbar_syn12 regulate the post-golgi transport of aquaporin PIP2-7 to the plasma membrane, thereby regulating cell membrane permeability. Gbar_A05G000580 (Gbar_4CL 1) is located at a key position in the phenylpropane metabolic pathway, controlling the synthesis of flavonoids and lignin. Gbar_A05G000660 (Gbar_MYB36) and PR1 are combined to regulate drought resistance and verticillium wilt resistance of cotton.

(2) Fine localization and map-bit cloning of gbar—4cl1: the genotype data of the F _2:3 group is analyzed by QTL ICIMAPPING 4.0.0 software, 11 candidate genes exist in combination with the phenotype data preliminary localization interval of F ₂, 30 recombinant single plants of the screened F ₂ group are planted into F _2:3 strains, each single plant is not less than 50 single plants, total 1500 single plants, disease resistance investigation is carried out on the recombinant single plants in the ten days of 8 months of 2023, fine localization analysis is carried out by using the same software, and a very high LOD peak value is excited between markers K183 and K191(K183_F:5'AGATACAAAGAGTAAGGGATAGGG 3'、K183_R:5'TGACTTGAATGATAGTAGTT AAAGTTCT 3';K191_F:5'TCTTTTTACGAATATGCTAGCTAAA 3'、K191_R:5'ATTCCAAAAA AAATCAATTTGTTT 3') (figure 2D), wherein the reason is that the screened recombinant single plants are exchanged near the preliminary localization interval. The final remaining candidate genes were: gbar_A05G000560 Gbar_A05G000570 Gbar_A05G000580 and Gbar_A05G000590.

In order to further improve the accuracy of the positioning result, the genotype and phenotype data are manually analyzed to finally see whether the positioning result can be consistent with the fine positioning of the software. By 9C 1 recombination type plants, the disease resistance of the type plants is weak as a whole and is positioned at the 3 rd level of disease resistance, so that the target gene should not be between K130 and K175 and should be after K175. The recombination type of C3 has stronger overall disease resistance at the 1 st level of disease resistance, and can indicate that the target gene is not between the K191 and the K215 markers. In combination with C7, C8 and C9, the target gene interval is at least between K175 and K191, so that the description accords with the algorithm expectation of software, and the software result is reliable (figure 2E).

The comparison was performed using (Li et al 2023.Comparative transcriptome analysis of interspecific CSSLs reveals candidate genes and pathways involved in verticillium wilt resistance in cotton(Gossypium hirsutum L.).Indus trial Crops&Products,2023,197) transcriptome data (the former was used as a reference genome for upland cotton at university of martial arts, while focusing on only high differential expression genes, and this analysis considered low differential expression genes simultaneously) with the whole genome sequence of TM-1 at university of agriculture in chinese and the gene annotation file. The difference genes were found by performing pairwise comparisons with Deseq (4.2), the parameters were set to Padj <0.05, |log FoldChange | >1. The comparison group ：E22 0H VS E22 24H、E22 0H VS E22 48H、E22 0H VS E22 72H、M34 0H VS M34 24H、M34 0H VS M34 48H、M34 0H VS M34 72H、E22 0H VS M34 0H、E22 24H VS M34 24H、E22 48H VS M34 48H、E22 72H VS M34 72H. was set up using the R language (4.2.3), and volcanic mapping was performed to look at the number of differential genes found for each comparison group, which were 1869, 4476, 3679, 5928, 1967, 4439, 2258, 4162, 5119 and 1640, respectively. Drawing a wien diagram (fig. 2F) using R language, finding the differential genes (genes within the candidate interval) present in each comparison group, finding that the differential genes present in the candidate interval are respectively: gbar_A05G000520 Gbar_A05G000580 Gbar_A05G000600 Gbar_A05G000660. The found 4 different genes are subjected to volcanic drawing by using the expression quantity of transcriptome data, and the expression quantity change of the 4 genes in different time periods after inoculation is observed (figure 2G), so that the expression quantity of Gbar_A05G000580 is obviously larger in M34 than in E22, and the expression quantity trend in the two genes takes 24H as an inflection point, and rises and then falls. The expression level of Gbar_A05G000660 gene E22 is slightly higher than M34, and the inflection points of the overall trend are inconsistent, wherein the former reaches a peak value at 24H, while the latter is at 48H, and the overall trend is rising and then falling. The Gbar_A05G000520 gene expression is slightly higher than M34, wherein the E22 reaches a peak value at 24H, and then rises and falls, and the E22 reaches a minimum value at 48H, and then falls and rises. The expression quantity E22 of Gbar_A05G00060 gene in two parents is slightly higher than M34, the former is descending, ascending and descending expression rules are not very fixed in four time periods, and the latter is firstly descending and then ascending at 24H as an inflection point. By analysis, the expression trend of Gbar_A05G000580 (Gbar_4CL 1) is more expected, the gene reaches a peak at 24H in response to V991 infection, and the expression level of the gene at M34 is higher than that of E22. In summary, gbar_4CL1 was finally determined as the target gene.

Example 3Gbar_4CL1 Gene responds to verticillium infection at E22 and M34

(1) Plant material sources and planting methods: selecting 200 large and full E22 and M34 seeds respectively, accelerating germination, transferring to 1/2MS nutrient solution, transferring to a climatic chamber (28 ℃ C., 16H light and 8H dark) for culture, and balancing for 4-5 days (the specific planting method is the same as that of embodiment 1).

(2) Activation, culture, inoculation and disease finger statistics of verticillium wilt bacteria: the specific operation method is the same as in embodiment 1.

(3) RNA extraction and expression level detection: root RNAs of E22 and M34 were extracted from the inoculated strain V991 and the non-inoculated strain V991H, 4H, 8H, 12H, 24H, 48H and 72H, and the trend of the variation in the expression level of the gene was observed.

(4) Results analysis of gbar—4cl1 for response V991 infestation: in order to identify the dominant expression site of Gbar_4CL1, the tissue expression mode of the gene is obtained, RNA of root, stem and leaf tissues of two-leaf one-heart parents E22 and M34 is extracted, and the difference of the expression amounts of different tissues is observed, and the result is shown in FIG. 3A. As is clear from FIG. 3A, the expression level in the stem is highest, followed by root and leaf, the expression level of the gene in M34 is about 1.2, the expression level of E22 is about 0.7, and the expression level of M34 is significantly higher than E22. The purpose of the tissue expression pattern was to examine whether the gene expression level E22 was also smaller than M34 at the early stage of cotton seedling, and whether V991 caused a difference in the expression levels of the two parents, and as a result, as shown in FIG. 3A, gbar-4 CL1 was significantly higher than E22 even if it was not infected with V991.

To verify if gbar_4cl1 would respond to infection by verticillium, root RNAs of E22 and M34 in the presence of and absence of inoculum V991H, 4H, 8H, 12H, 24H, 48H, 72H were extracted, and the response of the gene was examined by observing the trend of variation in the expression level of the gene, as a result, see fig. 3B and 3C. As can be seen from FIGS. 3B and 3C, the expression level of Gbar_4CL1 in the range of 0 to 24H was gradually increased, the expression level of 24 to 72H was gradually decreased, and the gene was not significantly changed in the control group without inoculation, and it was found that the expression level was higher in each period after inoculation than in the absence of inoculation by comparing each period of inoculation with that without inoculation. Taken together, gbar—4cl1 was shown to be induced by verticillium.

Example 4Gbar_4CL1 sequence difference between the promoters of E22 and M34 and CDS region

(1) Plant material sources and planting methods: 20 large and full E22 and M34 seeds are selected, germinated, transferred to 1/2MS nutrient solution, transferred to a climatic chamber (28 ℃ C., 16H light and 8H dark) for cultivation, and balanced for 4-5 days (the specific planting method is the same as that of embodiment 1).

(2) Extraction of E22 and M34 DNA: extracting E22 and M34, placing about 0.1g of tender leaves in a centrifuge tube with a heart period of about 2mL into an ice box or liquid nitrogen, placing the tender leaves on ice, adding steel balls and 200 mu L of extraction buffer solution one by one after the swing on the ice, and beating a sample grinder for 60s at the frequency of 60Hz; at the moment, the water bath kettle can be adjusted to 65 ℃; adding 800 mu L of preheated DNA extraction lysate, and shaking uniformly to ensure that the sample and the lysate are fully and uniformly contacted; water bath at 65 ℃ for 30min, gently reversing for several times every 10min, and avoiding violent oscillation in the subsequent steps; adding 800 μl of chloroform, gently shaking, extracting for 20min, and centrifuging at 11000rpm at room temperature for 10min; at the moment, the centrifugal rotating speed of the strip steel beads cannot be too high; sucking the supernatant to a new tube, adding isopropyl alcohol with equal volume, and uniformly mixing, wherein white flocculent precipitate is generated at the moment to obtain a DNA crude extract; pouring out the supernatant, washing the flocculent precipitate twice with 75% alcohol, drying, and dissolving with a proper amount of ddH ₂ O; in general, only 20-100ng of DNA is required for the template in PCR.

(3) Extraction of RNA of E22 and M34: RNA was extracted from root, stem and leaf tissues of the two-leaf-heart stage parent E22 and M34 (3 replicates per tissue).

(4) True sequence amplification of gbar_4cl1 at the promoters and CDS regions of E22 and M34: the promoter of Gbar_4CL1 and the primer of the CDS region are designed by using (https:// cottonfgd.net /) websites and taking cotton TM-1 at agricultural university in China as a reference genome, and amplification and sequencing are carried out at the same time, wherein the primers are as follows:

CDS-Gbar_4CL1_F：ATGGAGGCCGATCAACACCAGC

CDS-Gbar_4CL1_R：CTAGTTGGCTACATCTGCAGCGAGC

PRO-Gbar_4CL1_F：CTTCATCACTTGATTGCATGAATAT

PRO-Gbar_4CL1_R：ACATTGGCTATGTTGTTGAATCCCT

(5) Analysis of the results of sequence differences between the promoters of gbar_4cl1 and the CDS region at E22 and M34: the results of the comparison and analysis of the sequencing data are shown in FIG. 4.

The nucleotide sequence of Gbar_4CL1 in M34 (shown as SEQ ID NO.1, wavy line indicates promoter, underlined indicates exon) is:

The CDS sequence of Gbar_4CL1 gene in M34 is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 3.

The nucleotide sequence of Gbar_4CL1 in E22 is shown in SEQ ID NO.4, and only the promoter sequence of Gbar_4CL1 in E22 is shown below:

the CDS sequence of Gbar_4CL1 gene in E22 is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6.

As can be seen from FIG. 4, the nucleotide sequences of the gene in the CDS regions of the two parents show that only one nucleotide change (T and A transversions) exists in the CDS regions, and phenylalanine is changed into tyrosine. And the promoter sequences of E22 and M34 have base deletions at 800bp, 752bp and 150bp, and simultaneously have base substitutions at a plurality of positions. By combining the expression amounts of the Gbar_4CL1 genes in FIG. 3B and FIG. 3C in different materials, it can be determined that the base change of the promoter region affects the expression of the Gbar_4CL1 genes in E22 and M34, thereby causing the phenotype difference of E22 and M34. The Gbar_4CL1 gene in M34 is naturally expressed in high level, so that the disease resistance is obviously improved.

Example 5gbar_4cl1 silencing in cotton

(1) Construction of VIGS vector: according to the nucleic acid sequence information of the CDS region of a candidate genome candidate gene of TM-1 of cottonfgd (https:// cottonfgd.net /), carrying out blastn comparison, taking a specific section of a target gene as a reference sequence, designing a primer, controlling the target fragment to be 300-500 bp, adding a Kpn I restriction site adaptor Kpn I-adapter (5'-GCGTGAGCTCGGTACC-3') at the 5 'end of a forward primer, adding a Bam HI restriction site adaptor Bam HI-adpter (5'-GCCTCCATGGGGATCC-3') at the 5' end of a reverse primer, amplifying by taking cDNA of a material M34 stalk as a template, and purifying a PCR product after detecting that the size of the PCR product is correct by electrophoresis, wherein the primer sequence is as follows:

VIGS-Gbar_4CL1_F：AATTCTCTAGTTTGAGAATGTTTCCCAGTTTAAAG；

VIGS-Gbar_4CL1_R：GTACCGGATCGAAACTCTTCACTTTCTCCGCAT。

Purified PCR product and TRV after BamHI and KpnI double cleavage with In Fusion (Clonetch) enzyme: the 00 plasmid ligation was transformed into competent E.coli TOP10. The specific connection system is as follows: exase II 1.0uL, 5 XBuffer 2.0uL, double digested TRV:00 plasmid 100ng, purified PCR product 10ng, ddH ₂ O to 10uL. The prepared In Fusion system is subjected to water bath at 37 ℃ for 30min, ice bath for 5min, melting competent TOP10, ice bath for 20min, ice bath for 90s at 42 ℃, rapidly placed on ice, transferred to 200 mu L of LB culture medium after 2min, subjected to shaking at 180rpm for 40min at 37 ℃, 100 mu L of shaking bacteria are spread on a dish of LB solid culture medium (containing 0.1% kanamycin), placed on a constant temperature incubator at 37 ℃ for 12H In a reverse buckle mode, subjected to monoclonal selection, subjected to PCR amplification positive detection by using Gene-F and TRV:00-F primers, and subjected to sequencing of positive strains by biological companies. The correct monoclonal shaking bacteria are sequenced, plasmids are extracted, positive plasmids are transferred to competent agrobacterium GV3101 by an electric shock method, bacterial liquid is transferred to 2mL LB liquid culture medium after electric shock, a constant temperature shaking table at 180rpm at 28 ℃ is used for shaking 1H, 100 mu L of bacterial liquid is coated on solid LB culture medium containing 0.1% kanamycin and 0.1% rifampicin, the solid LB culture medium is placed in a constant temperature incubator at 28 ℃ in a reverse way for culturing for 2 days, and PCR positive detection is carried out by using Gene-F and TRV:00-F primers. The positive strain is stored in a refrigerator at-80 ℃ for standby by using glycerol to dilute the bacterial solution until the final concentration of the glycerol is 20%.

(2) Activation, culture, inoculation and disease finger statistics of verticillium wilt bacteria: the specific operation method is the same as in example 1.

(3) VIGS bacterial liquid operation and treatment: strain activation: 20. Mu.L of the constructed strain containing the desired fragment, the indication control strain TRV: CLA, the negative control strain TRV:00 and the auxiliary vector strain TRV1 were inoculated in 500. Mu.L of liquid LB medium (0.1% rif and 0.1% kan) (2 mL centrifuge tube) 1 day in the morning before VIGS treatment, activated and propagated at about 10H at 180rpm at 28℃on a shaker, and an appropriate amount of the activated strain was transferred to 5mL of liquid LB medium (containing 0.1% rif,0.1% kan) and propagated at 180rpm on a shaker overnight at 28 ℃. And (3) collecting and activating thalli: the overnight cultured bacterial liquid was transferred to a 10mL centrifuge tube and centrifuged at 4000rpm for 15min at room temperature. The supernatant was discarded, resuspended in suspension (final concentration 200. Mu.M acetosyringone, 10mM MES,10mM MgCl2), the absorbance of each bacterial liquid OD 600 was measured, OD 600 was adjusted to 0.6-0.8, the bacterial cells were mixed with TRV1 in equal volumes, and the cells were shaken at 28℃and 180rpm for 1H or left at room temperature for 3-5H. VIGS processing: discarding weak seedlings, selecting cotton seedlings with thick cotyledons and consistent growth vigor, lightly screwing scratches on the back surfaces of the cotton cotyledons by using a 1mL pipette tip (note that the cotyledons cannot be penetrated), uniformly filling thallus suspension on the back surfaces of the leaves at the positions of the scratches by using a 1mL syringe for removing the needle, coating a black plastic film after treatment for preventing excessive water loss of the cotton seedlings after treatment and reducing treatment harm, removing the film after the darkness treatment for 24H, and culturing in a climatic chamber at 25 ℃ under 16H light/8H darkness.

(4) And (3) silencing effect detection: the actual leaf of the control material TRV: CLA was indicated to exhibit a pronounced albino phenotype, typically 10-14 days after treatment. When the TRV is subjected to CLA treatment and the true leaves have obvious albino phenotype, respectively taking 5 leaves or stems (corresponding parts of TRV: CLA material albino) subjected to silencing treatment and negative control TRV:00, rapidly placing the leaves or stems in liquid nitrogen, transferring the liquid nitrogen to a refrigerator at-80 ℃ for short time for standby, extracting and reverse transcription of RNA, detecting the expression quantity of a target gene by qRT-PCR, and compared with the negative control TRV:00, evaluating the silencing efficiency of the target gene, wherein the silencing effect is obvious, and then the subsequent test can be performed.

Reverse transcription specifically comprises the steps of: after RNA extraction, the quality of RNA was detected by agarose gel electrophoresis, the concentration of RNA was detected by a NanoDrop 2000 micro-spectrophotometer, 3. Mu.g of RNA was reverse transcribed into cDNA, and the reverse transcription reagent was PROMEGA company reagent. 3. Mu.g RNA, 1. Mu.L oligo (dT), was added to a 0.5mL enzyme-free centrifuge tube, DEPC water was added to 15. Mu.L, and after mixing, the mixture was placed on a reverse transcriptase for 5min at 70℃and then on ice for 10min. 5μL 5×MLV Buffer,1.25μL 10mmol/L dNTPs,1μL Rnasin(40U),1μL M-MLV RTase(200U),1.75μL RNase free H₂O, total volume was added in proportion to 25. Mu.L. Mixing, placing in a reverse transcription instrument at 42deg.C for 60min and at 70deg.C for 15min, diluting the reverse transcribed cDNA with ddH ₂ O to 250 μl, and storing at-20deg.C. The cDNA was then diluted 10-fold as a template for subsequent expression analysis.

Specific procedure for qRT-PCR: 7.5. Mu.L of cDNA template, 7. Mu.L of SybrGreen-mix (BIO-RAD Co.), 0.25. Mu.L of forward and reverse primers, were mixed and placed in a real-time fluorescent quantitative PCR apparatus (ABIPrism 7500 system) with the procedure set up: stage1, 95℃30s,1 cycle; stage2, 95℃5s+60℃35s,40 cycles. The fluorescence intensity values were read at the end of each cycle, and the relative expression levels of the genes were calculated using the 2 ^-ΔΔCT method, ghUBQ7 (Ghir _a11g 011460) was used as an internal gene and the significance of differences in expression levels in the different materials of the genes was determined by t-test.

(5) Recovery culture of verticillium: taking the stalks of the same part (hypocotyl/first section) of cotton seedlings about 16 days after inoculation, diluting 84 disinfectant (luwa, available chlorine content 8000 mg/L-10000 mg/L) with sterilized water in an equal volume for 1 time, immersing the stalks in the diluted disinfectant for 5min, slightly shaking the disinfectant during the period, rinsing the excessive disinfectant on the surfaces of the stalks with sterile distilled water for 3-4 times, placing the stalks on sterilized filter paper in the ultra-clean workbench, cutting the stalks into 2-3 mm-sized stalks by a sterile cutter after the stalks are dried, placing the cut stalks on PDA culture medium according to material partition, inverting the culture in a culture box at 25 ℃ for 2-4 days, observing the growth condition of hyphae, and photographing.

(6) Verticillium bacteria-containing amount measurement: about 16 days after inoculation, taking one section of the inoculated cotton Miao Di, rapidly placing the inoculated cotton into liquid nitrogen, and preserving the inoculated cotton in a refrigerator at the temperature of minus 80 ℃ for later use. After the liquid nitrogen is fully ground, DNA is extracted by a DNA extraction kit (FastPure Plant DNA Isolation Mini Kit, vazyme, DC401-01, application method see product instruction book), the template is diluted to 10 ng/mu L to be used as the template after the concentration is measured, ghUBQ (Ghir _A11G 011460) is used as an internal reference gene, ITS1-F and ST-VE1-R are verticillium fungus specific primers, and a qRT-PCR method is used for detecting fungus biomass in the stems.

(7) Preparation of split bars for VIGS experimental group and negative control group: in the silencing effect detection, the stems of the plant experimental group and the control group (on day 14 after V991 infection) were randomly excised, 3 replicates of each group were taken, and photographed.

(8) Analysis of results after silencing gbar—4cl1: gbar_4CL1 was silenced using the VIGS technique, while inoculation with V991 observed whether a phenotypic difference occurred in M34. When TRV: CLA developed a albino phenotype (about 10 days), the control TRV:00 was compared with the experimental TRV: gbar_4CL1, and the results are shown in FIGS. 5A and 5B. As can be seen from fig. 5A and 5B, the control group showed fallen leaves, partial yellow and wilted states, whereas the experimental group showed fallen leaves, yellow and wilted states in a large area, and the phenotype difference was obvious. Silencing effects of gbar_4cl1 were investigated with disease fingers and the results are shown in fig. 5C and 5D. As can be seen from FIGS. 5C and 5D, the Gbar_4CL1 gene silencing effect reached 50%, indicating that the gene silencing effect was good. After silencing the gene, the two groups of disease fingers TRV 00 and TRV Gbar_4CL1 show significant differences, and the disease finger of M34 is increased by 3 times. After 3 days of stem recovery culture in a biochemical incubator, the recovery culture condition and the stem recovery culture bacteria content were investigated, and the results are shown in fig. 5E and 5F. As can be seen from FIGS. 5E and 5F, a small amount of diseased stems appear in the stem of TRV 00, and the diseased stems of TRV Gbar_4CL1 are in most part, the relative bacterial content of TRV 00 is about 0.35 after the detection of the relative bacterial content, and the bacterial content of TRV Gbar_4CL1 reaches about 1.25, so that the bacterial content is obviously different. On day 14 after V991 infestation, the same-position stalks of TRV 00 and TRV Gbar_4CL1 were cut out, and the longitudinal sections were cut out with a knife, with the result shown in FIG. 5G. As can be seen from FIG. 5G, the cross-sectional bar of TRV Gbar_4CL1 was observed to have severe blackening and browning, whereas the onset of the control group was lighter.

In summary, M34 disease resistance was greatly reduced after silencing gbar—4cl1.

Example 6 silencing gbar_4cl1 disrupts lignin deposition, reducing cotton verticillium resistance

(1) The material sources are as follows: the stems of the negative control TRV 00 and the experimental group TRV Gbar_4CL1 of example 3 were cut about 3cm (3 were randomly cut per group).

(2) Paraffin section observation lignin deposition: immersing the materials in 70% FAA fixing solution, then making slices, and concretely performing paraffin wax slicing: taking out a standby hypocotyl sample from a refrigerator at the temperature of 4 ℃, pouring out a fixing solution in a centrifuge tube, rinsing the sample for 2 times by using clear water, embedding stem sections in a 1.5mL centrifuge tube by using 4% -5% agarose, cooling to room temperature, completely solidifying the agarose, tearing the centrifuge tube by using a utility knife, carefully taking out an embedded block, cutting off superfluous agarose, firmly adhering the embedded block on a sample table of a vibrating microtome (Leica, VT 1000S) by using 502 glue after trimming, adjusting the positions of the sample table and a cutter head, fixing the sample table, setting parameters such as slice thickness (75-90 mu m), slice total thickness, slice vibration frequency and the like, starting slicing, and gently transferring the cut slice into the centrifuge tube filled with distilled water by using a thin and soft writing brush head. After slicing, the distilled water in the centrifuge tube is gently sucked out, 500 mu L of 3% phloroglucinol solution (dissolved by 100mL of 95% ethanol) is added for dyeing, after dyeing for 10min, the dyeing solution is discarded, a proper amount of 18% HCL (phloroglucinol can react with lignin in the cell wall in an acidic environment to display brick red or purplish red) is added for color development, the centrifuge tube is turned upside down for several times, the slices are quickly transferred to a glass slide after color development for about 30 seconds, and the glass slide is placed on a stereoscopic microscope (Nikon, SMZ 25)

(3) Analysis of lignin deposition in paraffin sections: on day 14 of infection of M34 with V991, the stems of TRV 00 and TRV Gbar_4CL1 were excised and stained with phloroglucinol-HCl, and the reagent combined with lignin to form pink organic material, from which lignin deposition was observed. Since the stem segments of the cut TRV 00 and TRV Gbar_4CL1 are derived from cotton seedlings of approximately 14 days or so of M34, the plant preferentially deposits lignin near the vascular bundles to survive the plant in order to maintain the supply of water and nutrients to the plant during the primary growth stage of the vascular bundles, and the vascular bundles are more deeply colored. As can be seen from FIG. 6, the vascular bundle red of the stem paraffin sections of TRV Gbar_4CL1 is lighter than the TRV 00 red. The gene is located in the upstream pathway of the phenylpropane metabolic pathway and determines the metabolism of flavonoids and lignin. Silencing Gbar_4CL1 gene destroys lignin metabolic pathway, resulting in less or even no accumulation of lignin and destruction of plant feedback regulation system, so that TRV Gbar_4CL1 has less accumulation of lignin in the vascular bundle and lighter red color.

Taken together, gbar_4cl1 is a key gene for lignin pathway metabolism, and silencing gbar_4cl1 lignin metabolic pathway is disrupted.

The above detailed description describes in detail the practice of the invention, but the invention is not limited to the specific details of the above embodiments. Many simple modifications and variations of the technical solution of the present invention are possible within the scope of the claims and technical idea of the present invention, which simple modifications are all within the scope of the present invention.

Claims (10)

1. A cotton Verticillium wilt resistance gene Gbar_4CL1, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1. 2. Products containing any of the following a1)-a3): a1) an expression cassette encoding the gene according to claim 1; a2) a recombinant vector containing the expression cassette described in a1); a3) A recombinant microorganism containing the expression cassette described in a1) or the recombinant vector described in a2). 3. Use of the gene according to claim 1 or the product according to claim 2 in enhancing the disease resistance of cotton, preparing a product for enhancing the disease resistance of cotton, or breeding disease-resistant cotton varieties. 4. The use according to claim 3, characterized in that the disease resistance is resistance to cotton Verticillium wilt. 5. A method for enhancing the disease resistance of cotton or cultivating disease-resistant cotton varieties, characterized in that the gene according to claim 1 or the product according to claim 2 is introduced into cotton for expression. 6. The method according to claim 5, characterized in that the disease resistance is resistance to cotton Verticillium wilt. 7. A method for cultivating transgenic plants with reduced resistance to Verticillium wilt, comprising the following steps: inhibiting the expression of the gene of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has lower resistance to Verticillium wilt than the recipient plant. 8. The method according to claim 7, characterized in that the resistance of the transgenic plant to Verticillium wilt is lower than that of the recipient plant as shown in the following b1) or b2): b1) The susceptibility of transgenic plants to disease is higher than that of recipient plants; b2) The disease susceptibility index of transgenic plants is higher than that of recipient plants. 9. The method according to any one of claims 7-8, characterized in that inhibiting the expression of the gene according to claim 1 in the recipient plant comprises: constructing a TRV:00 vector of the Gbar_4CL1 gene using VIGS technology to silence the Gbar_4CL1 gene in the recipient plant. 10. The method according to any one of claims 7-8, wherein the plant comprises cotton.