CN104829700A - Corn CCCH-type zinc finger protein, and encoding gene ZmC3H54 and application thereof - Google Patents

Abstract

The invention discloses a corn CCCH-type zinc finger protein and its coding gene ZmC3H54 and its application. The amino acid sequence of the corn CCCH-type zinc finger protein of the invention is shown in the sequence table SEQ ID NO.1. The coding gene ZmC3H54 of the maize CCCH-type zinc finger protein according to the present invention has a nucleotide sequence as shown in SEQ ID NO.2 of the sequence table. The invention contains the recombinant vector, recombinant bacterium, transgenic cell line or expression cassette of the coding gene ZmC3H54. The ZmC3H54 gene of the invention participates in stress response, and can improve the ability of plants to resist abiotic adversity stress. Transgenic plants have higher sensitivity to ABA, and transgenic rice has improved tolerance to drought. The present invention isolates and clones the family gene from maize and identifies its function in improving the stress resistance of target plants.

Description

A kind of corn CCCH type zinc finger protein and its coding gene ZmC3H54 and application

technical field

The invention belongs to the field of crop genetics and breeding, and in particular relates to a corn CCCH zinc finger protein, its coding gene ZmC3H54 and its application.

Background technique

Corn is an important food crop and an important source of feed, and it is also the food crop with the highest total output in the world. Corn is the most suitable variety among the three major grain crops as industrial raw materials, and occupies an important position in the national economy. However, abiotic stresses such as drought and high salinity seriously affect the yield of maize. Therefore, it is the key to realize the sustainable development of maize production to discover and create new germplasm of maize with strong stress resistance, high yield and high quality and to breed new varieties. With the rapid development of molecular biology and plant genetic engineering breeding technology, improving the productivity of maize under adverse conditions and cultivating new varieties with strong resistance through genetic engineering breeding technology has become a research hotspot in genetic improvement of stress resistance.

This CCCH zinc finger protein widely exists in eukaryotes and is an important regulatory factor. It plays a role that cannot be ignored in the growth and development of animals and plants and the response to external adversity. Most of them are related to RNA It is related to the metabolism of genes, and can usually regulate the expression of genes at the transcriptional level or the post-transcriptional level. There are many CCCH family genes in plants. In recent years, 68 and 67 CCCH family genes have been identified in two model plants, Arabidopsis thaliana and rice, through the method of bioinformatics. Most members of the family are related to the stress of Arabidopsis, which is the first step for the extensive study of this class of proteins in plants (Wang et al., 2008a).

Some CCCH zinc finger proteins have been proved to play a role in plant development, response to environmental stress, etc. through overexpression or inhibition of transgene and gene expression analysis. CCCH-type zinc finger protein is a kind of zinc finger protein family. A typical CCCH zinc finger protein contains at least one or more zinc finger motifs arranged in sequence of 3 Cys and 1 His. This structure can be Summarized as: CX _5-14 -CX _4-5 -CX ₃ -H. X stands for any amino acid residue, (Blackshear, 2002; Wang et al., 2008a).

For example, OsDOS was found to be associated with rice leaf senescence (Kong et al., 2006); AtSZF1 and AtSZF2 were found to play a role in the response of Arabidopsis to salt stress (Sun et al., 2007); GhZFP1 was found to be involved in tobacco Stress responses to salt and pathogens (Guo et al., 2009). AtTZF1 is a sugar-sensitive protein involved in gibberellin/abscisic acid-mediated plant development and responses to environmental stress (Pomeranz et al., 2010). The functional reports of these CCCH-type zinc finger protein-related stress resistance genes are mostly reported in Arabidopsis, rice, cotton, and few in maize. There have been many studies on abiotic stress. Abiotic stresses such as low temperature, high salinity, and drought generally cause some gene changes or the combination of some transcription factors.

At present, there is a lack of a maize CCCH-type zinc finger protein ZmC3H54 and its coding gene ZmC3H54 with strong resistance to abiotic stress.

Contents of the invention

The purpose of the present invention is to provide a maize CCCH type zinc finger protein with strong ability to resist abiotic adversity stress, its coding gene ZmC3H54 and its application.

In order to achieve the above objectives, the present invention is achieved through the following technical solutions: a corn CCCH-type zinc finger protein of the present invention, the amino acid sequence of which is shown in the sequence table SEQ ID NO.1.

The coding gene ZmC3H54 of the corn CCCH-type zinc finger protein according to the present invention has a nucleotide sequence as shown in SEQ ID NO.2 of the sequence table.

The invention contains the recombinant vector, recombinant bacterium and transgenic cell line containing the coding gene.

Application of the maize CCCH-type zinc finger protein or the coding gene ZmC3H54 of the maize CCCH-type zinc finger protein in the cultivation of transgenic plants resistant to abiotic stress.

Further, in the application, the transgenic plant is rice or Arabidopsis.

A method for preparing a transgenic plant resistant to abiotic stress of the present invention comprises the following steps:

(1) Obtain the nucleotide sequence and amino acid sequence of the coding gene ZmC3H54 of the corn CCCH-type zinc finger protein;

(2) obtain the corn ZmC3H54 gene fragment by PCR;

The expression profile of maize gene ZmC3H54 under adversity stress was analyzed by fluorescent quantitative PCR method, and the ZmC3H54 gene fragment was constructed into a plasmid vector;

(3) transforming the plasmid with ZmC3H54 obtained in step (2) into Agrobacterium;

Transforming the target plant with the Agrobacterium transforming plasmid;

(4) Screening and identification of transgenic positive seedlings of target plants; analysis of abiotic stress resistance of transgenic homozygous plants.

Further, in step (1), after the cloning vector plasmid containing the ZmC3H54 gene is double digested by BamHI+XbaI, a DNA recovery kit is used to reclaim the 1119bp DNA fragment of SEQ ID NO.2, and this fragment is combined with the corresponding enzyme The pCAMBIA1301a vector was cut and connected, and the obtained vector was named p1301a-ZmC3H54 recombinant vector;

The primer sequence of the gene is cloned from corn cDNA as a template PCR, and the primer of the amplified ZmC3H54 gene is divided into an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in the sequence table SEQ ID NO.5 , the nucleotide sequence of the downstream primer is shown in the sequence table SEQ ID NO.6;

In step (3), the target plant is rice or Arabidopsis.

In step (4), the abiotic stress resistance analysis includes drought tolerance analysis and sensitivity analysis of plant seed germination to plant hormone ABA.

A method of the present invention for in vitro detection of ZmC3H54 gene expression in plants after induction and expression differences in various tissues in plants, comprising the following steps:

(1) Take the leaf treatment samples and several different tissue samples of the plants at various time periods after the corresponding induction of the plants;

(2) Extract the RNA of each sample with the Trizol method;

(3) Digest the DNA with DnaseI, then reverse transcribe to form cDNA, and use it as a template;

(4) Analyze with fluorescent quantitative PCR, the detection primer sequence of fluorescent quantitative PCR, described primer is divided into upstream primer and downstream primer;

The nucleotide sequence of the upstream primer is shown in the sequence listing SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in the sequence listing SEQ ID NO.4.

Further, in step (1), the plant is corn;

Stress treatment sampling: the plant material is corn B73 inbred line. When the corn seedlings grow to the three-leaf stage, the normal irrigated plants are used as the control, and the stress treatments of 10% PEG solution, 100uM ABA solution and 200mmol/L NaCl solution are respectively carried out , samples were taken after treatment for different time periods, the time was 0h, 1h, 3h, 6h, 12h, 24h respectively; the leaves of the same part of the corn seedlings of the test group and the control group were cut with scissors, and three samples were taken at each treatment stage; , and the samples were stored frozen in liquid nitrogen at -80°C;

Tissue sampling: In order to analyze the difference in the expression level of the maize ZmC3H54 gene in various tissues, 8 representative tissues of maize were selected, and the representative tissues were root, stem, young leaf, silk, tassel, pre-pollination Ears, bracts, embryos and endosperms were sampled and stored in liquid nitrogen at -80°C;

In step (4), the detection of total RNA purity and content: take 1 μ L of total RNA sample and add it to the micro-spectrophotometer probe to detect the total RNA purity and content;

Get normal corn as a contrast, corn internal reference gene Actin primer, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, the nucleotide sequence of described downstream primer The nucleotide sequence is shown in the sequence listing SEQ ID NO.8.

Further, in step (4), normal Arabidopsis is taken as a contrast, and the Arabidopsis internal reference gene Actin primer is divided into an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as follows: As shown in the list SEQ ID NO.9, the nucleotide sequence of the downstream primer is shown in the sequence table SEQ ID NO.10.

Beneficial effects: the ZmC3H54 gene of the invention participates in stress response, and can improve the ability of plants to resist abiotic stress. Both transgenic rice and transgenic Arabidopsis have high sensitivity to ABA, and the tolerance of transgenic rice to drought is improved. The present invention isolates and clones this family gene from corn and identifies its role in improving the stress resistance of target plants. It is of great significance for the cultivation of stress-resistant new crop varieties.

Compared with the prior art, the present invention has the following advantages:

(1) The present invention isolates and clones the ZmC3H54 gene from corn, and the induced expression analysis finds that the expression of ZmC3H54 is induced by exogenous ABA, PEG and high salt, and it is introduced into rice and Arabidopsis respectively through the transformation method mediated by Agrobacterium mustard and obtain the corresponding transgenic plants. Transgenic plants have increased sensitivity to ABA and enhanced drought tolerance.

(2) The present invention is based on bioinformatics analysis, and according to its evolutionary relationship and stress-induced expression pattern, a gene encoding a CCCH-type zinc finger protein that is strongly induced by drought and ABA is screened, and its expression is regulated by transgenic technology. The expression level in the plant can improve the stress resistance of the plant. In the present invention, a semi-quantitative PCR method can be used to analyze the expression level of the ZmC3H54 gene in the transgenic Arabidopsis. Specifically, primers were designed according to the sequence of the transcription factor ZmC3H54 gene, and the ZmC3H54 gene was amplified from the total cDNA of the maize inbred line B73 by using PCR technology. Induced expression analysis found that the expression of ZmC3H54 was induced by exogenous ABA, PEG and high salt.

(3) The present invention provides important genetic resources for plant stress-resistant genetic engineering, is beneficial to the research of new stress-resistant varieties, and is of great significance for improving crop yield.

Description of drawings

Figure 1 is a comparison diagram of amino acid sequence homology between ZmC3H54 of the present invention and the conserved regions of CCCH family zinc finger proteins of other species;

Fig. 2 is the phylogenetic tree after the amino acid sequence alignment of ZmC3H54 of the present invention and the CCCH zinc finger protein family with reported functions in Arabidopsis and other plants;

Fig. 3 is the induced expression pattern analysis of the ZmC3H54 gene of the present invention when treated with ABA, drought, and high-salt stress and the tissue expression pattern diagram of the ZmC3H54 gene; A: 10% PEG solution treatment; B: 100uM ABA solution treatment; C: 200mmol/LNaCl solution treatment;

Fig. 4 is the comparison chart of the improvement of the tolerance of the transgenic ZmC3H54 rice of the present invention to drought; A: 15 days of drought treatment; B: 30 days of drought treatment; C: recovery of watering for 20 days; D: wild growth after recovery of 20 days type and survival rate of transgenic plants;

Fig. 5 is the comparison chart of the improvement of the sensitivity of transgenic ZmC3H54 rice of the present invention to ABA; A-C: the growth phenotype, seedling height and root length of wild-type and transgenic seedlings in the MS nutrient solution of different concentrations of ABA;

Fig. 6 is the expression analysis diagram of ZmC3H54 of the present invention in wild-type (WT) Arabidopsis and T1 generation transgenic plants; A: fluorescence quantitative analysis; B: semi-quantitative analysis;

Fig. 7 is the comparison chart of the improvement of ABA sensitivity of transgenic Arabidopsis thaliana of the present invention; A: phenotype on MS medium; B: phenotype on MS+0.2ABA medium; C MS+0.4ABA medium The phenotype on the plate; D: the germination rate on the three plates;

Fig. 8 is a comparison diagram of the pCAMBIA1301 vector before transformation and the pCAMBIA1301a vector after transformation involved in the present invention.

Detailed ways

The present invention will be further described in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The methods used in the examples are conventional methods unless otherwise specified, and the primers used are all synthesized by Shanghai Sangon Biotechnology Co., Ltd.; the sequencing is carried out by Invitrogen; various restriction enzymes, ligases, and DNA Markers used in the experiments , Taq DNA polymerase, dNTPs, etc. were purchased from Takara Company; reverse transcription kits were purchased from Promega Company; plasmid extraction kits, gel recovery kits and genome extraction kits were purchased from Quanshijin Biotechnology Co., Ltd. Follow the instruction manual.

A corn CCCH-type zinc finger protein of the present invention has an amino acid sequence as shown in SEQ ID NO.1 of the sequence table.

The 44th-95th amino acid residue sequence from the amino terminal of the sequence listing SEQ ID NO.1 is a conserved structure of two typical CCCH-type zinc fingers CX ₈ CX ₅ CX ₃ H and CX ₅ CX ₄ CX ₃ H.

The nucleotide sequence of the gene encoding the maize CCCH-type zinc finger protein is shown in SEQ ID NO.2 of the sequence table. The present invention provides a nucleotide sequence of a new maize zinc finger protein coding gene, which codes a ZmC3H54 gene expressed induced by drought and ABA, GRMZM2G117007, mainly manifested in that the germination of its corresponding transgenic seeds is highly sensitive to ABA The plants have certain drought resistance. Maize C3H54 gene, GRMZM2G117007 full length 1466bp, cDNA coding region sequence length 1119bp, as shown in SEQ ID NO.2, this gene encodes 372 amino acids, and contains two typical CCCH-type zinc finger structures CX ₈ CX ₅ CX ₃ H and CX ₅ CX ₄ CX ₃ H, as shown in SEQ ID NO.1.

The coding gene ZmC3H54 of the corn CCCH-type zinc finger protein according to the present invention has a nucleotide sequence as shown in SEQ ID NO.2 of the sequence table.

The invention contains the recombinant vector, recombinant bacteria and transgenic cell line of the coding gene ZmC3H54.

Application of the maize CCCH-type zinc finger protein or the coding gene ZmC3H54 of the maize CCCH-type zinc finger protein in the cultivation of transgenic plants resistant to abiotic stress.

In the application, the transgenic plant is rice or Arabidopsis.

Example 1

Acquisition of Maize ZmC3H54 Gene

After the corn seedlings grew to the three-leaf stage, total RNA was extracted and reverse transcribed into cDNA. The MAIZEGENOME and NCBI databases were searched to obtain the deduced coding sequence of ZmC3H54, and Primer Premier 5.0 software was used to design specific primers, which were synthesized by Sangon Biotech. The primer sequences are as follows:

ZmC3H54-F 5′-CG GGATCC ATGTACAACTTCAAAGTGAAGC-3′SEQ ID NO.5

BamHI

ZmC3H54-R 5′-GC TCTAGA TCAGGCCACCATCTGCTC-3′SEQ ID NO.6

XbaI

Using the total maize cDNA obtained above as a template, a coding region containing a complete open reading frame with a length of 1119 bp was obtained by PCR, recovered, connected to the pEASY-T1 vector, and sequenced. The obtained sequence is the maize ZmC3H54 gene.

Transformation of Plant Expression Vector pCAMBIA1301

The plant expression vector pCAMBIA1301 was transformed into a pCAMBIA1301a vector. FIG. 8 is a map of the pCAMBIA1301 vector before transformation and the pCAMBIA1301a vector after transformation involved in the present invention.

As shown in Figure 8, the specific steps are as follows:

(1) Use restriction endonucleases EcoRI and SacI to double digest and connect with T4 ligase, add a 35S fragment at the multiple cloning site of the pCAMBIA1301 vector, the direction is consistent with the GUS direction, and construct the pCAMBIA1301-35S vector;

(2) followed by double enzyme digestion with PstI and HindIII, and then ligated with T4 ligase, followed by adding a small segment of polyA to the 35S fragment to construct pCAMBIA1301-35S-polyA vector (hereinafter referred to as pCAMBIA1301a vector).

Construction of Maize ZmC3H54 Expression Vector

The obtained ZmC3H54+pEASY-T1 vector and pCAMBIA1301a vector were simultaneously digested with BamHI and XbaI. The enzyme digestion system is shown in Table 1:

Table 1

Add samples according to the double enzyme digestion system above, and recover the target fragment and the large fragment of pCAMBIA1301a vector after digestion for 3 hours. Then the ZmC3H54 target gene fragment recovered by enzyme digestion and the large fragment of the pCAMBIA1301a vector were ligated, and the ligation reaction system was shown in Table 2.

Table 2

After connecting for 3-5 hours at 16°C, the p1301a-ZmC3H54 recombinant vector was obtained after a series of transformation and verification, which is the expression vector of ZmC3H54.

Agrobacterium Transformation of Maize ZmC3H54 Expression Vector

The pCAMBIA1301-35S-ZmC3H54-polyA vector constructed and verified above was introduced into Agrobacterium EH105 by the freeze-thaw method. The specific steps are as follows:

(1) Take out Agrobacterium competent cells from -80℃ ultra-low temperature refrigerator, thaw on ice, add 3-5 μL of overexpression vector plasmid, flick and mix well, and place on ice for 5 minutes;

(2) Immediately freeze in liquid nitrogen for 1 minute, and quickly place in a water bath at 37°C for 5 minutes for heat shock;

(3) Add 300-500 μL of antibiotic-free YEP liquid medium, place in a shaker at 28°C, and culture at 220rpm for 5 hours;

(4) Centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, add 100 μL of resuspended bacteria, and spread on YEP solids containing 50 mg/L rifamycin (Rif) and 50 mg/L kanamycin (Kan). On the plate, after sealing, culture it upside down at 28°C for 2-3 days.

(5) Take a single colony and shake the bacteria, keep the bacteria, extract the plasmid, and then perform colony PCR or enzyme digestion verification.

(6) Store the verified bacterial solution at -80°C for later use.

Example 2

Expression pattern analysis and tissue pattern analysis of maize ZmC3H54 gene under PEG, ABA, NaCl conditions

As shown in Figure 3, it is the analysis of the induced expression pattern of the ZmC3H54 gene of the present invention and the tissue expression pattern of the ZmC3H54 gene during ABA, drought, and high-salt stress treatment; A: 10% PEG solution treatment; B: 100uM ABA solution treatment ; C: 200mmol/L NaCl solution treatment;

1. Stress treatment sampling: the plant material is the corn B73 inbred line. When the corn seedlings grow to the three-leaf stage, the plants that are normally irrigated are used as a control, and 10% PEG solution, 100uM ABA solution and 200mmol/L NaCl solution are used respectively. For stress treatment, samples were taken after treatment for different time periods, the times were 0h, 1h, 3h, 6h, 12h, and 24h; the leaves of the same part of the corn seedlings in the test group and the control group were cut with scissors, and three samples were taken at each treatment stage. After sampling, the samples were immediately frozen with liquid nitrogen and stored in a -80°C refrigerator;

Tissue sampling: In order to analyze the difference in the expression level of the maize ZmC3H54 gene in various tissues, 8 representative tissues of maize were selected, and the representative tissues were root, stem, young leaf, silk, tassel, pre-pollination Ears, bracts, embryos and endosperms were immediately frozen with liquid nitrogen and stored in a -80°C refrigerator after sampling;

2. Use the Trizol method to extract the RNA of each sample;

RNA extraction: total RNA was extracted according to the modified Trizol method, the specific steps are as follows:

(1) Take out the material from the -80°C refrigerator, put it into the grinder, put on gloves, add liquid nitrogen to grind, then transfer about 100mg of powder to a 1.5mL centrifuge tube, and pre-cool it in liquid nitrogen;

(2) Add 1mL Trizol reagent, mix vigorously up and down for 15s, and place at room temperature for 10min; centrifuge at 13,000rpm for 20min at 4°C;

(3) Take the supernatant into a new centrifuge tube, add 250 μL of 5M NaCl and 250 μL of chloroform; centrifuge at 10,000 rpm for 20 min at 4°C;

(4) Pipette the supernatant into a new centrifuge tube, add 200 μL of isopropanol, and place at room temperature for 10 minutes; centrifuge at 13,000 rpm for 15 minutes at 4°C;

(5) Carefully discard the supernatant, add 500 μL of 75% ethanol, and use DEPC water to prepare ethanol; centrifuge at 7,500 rpm for 5 min at 4°C, and pour off the ethanol;

(6) Repeat step (5), centrifuge at 7,500 rpm for 5 minutes at 4°C, pour off the ethanol, put it on paper, and let it dry at room temperature for 5-10 minutes;

(7) Shake the tube wall liquid to the bottom of the tube, store it at -70°C and take 2 μL of the test result.

3. Digest the DNA with DNaseI, then reverse transcribe to form cDNA, and use it as a template;

4. Detection of the purity and content of the total RNA: Take 1 μL of the total RNA sample and add it to the micro-spectrophotometer probe to detect the purity and content of the total RNA. A ₂₆₀ /A ₂₈₀ between 1.8-2.0 indicates that the quality of the extracted total RNA is high.

Analyze with fluorescent quantitative PCR, the detection primer sequence of fluorescent quantitative PCR, described primer is divided into upstream primer and downstream primer;

The nucleotide sequence of the upstream primer is shown in the sequence listing SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in the sequence listing SEQ ID NO.4.

Get normal corn as a contrast, corn internal reference gene Actin primer, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, the nucleotide sequence of described downstream primer The nucleotide sequence is shown in the sequence listing SEQ ID NO.8.

5. Removal of Genomic DNA Processing

(1) First, according to the RNA concentration of each sample detected in the previous step, calculate the amount of RNA to be added to each sample when the final concentration is unified. In the microcentrifuge tube, the reaction system is shown in Table 3, and the following are added in turn Reagent:

table 3

(2) After inching and mixing, incubate at 42°C for 2 minutes or at room temperature for 30 minutes before use.

6. Reverse transcription: operate according to the steps provided by the reverse transcription kit of Bao Bio Company, the reverse transcription system is as follows:

(1) In a microcentrifuge tube, the reverse transcription reaction system is shown in Table 4, and the following reagents are added in sequence:

Table 4

(2) After inching and mixing, keep warm at 37°C for 15 minutes;

(3) After treatment at 85°C for 5 minutes, after incubation at 4°C for several minutes;

(4) Centrifuge at room temperature for 5 seconds so as to collect all the solution to the bottom of the tube, dilute 10 times with RNase Free dH ₂ O and store in a -20°C refrigerator until use.

7. Fluorescent quantitative PCR reaction

(1) Primer design

Primers were designed using Primer Express 3.0 (ABI) software and synthesized by Shanghai Bioengineering Company.

The nucleotide sequence of the target gene ZmC3H54 detection primer is:

qPCR-ZmC3H54-F 5′-GGACGCGCCTCTGCAAG-3′SEQ ID NO.3

qPCR-ZmC3H54-R 5′-ATCTGCATGCCGACGGACA-3′SEQ ID NO.4

The nucleotide sequence of the maize internal reference gene Actin primer is:

qPCR-ZmActin-F 5′-GGGATTGCCGATCGTATGAG-3′SEQ ID NO.7

qPCR-ZmActin-R 5′-GAGCCACCGATCCAGACACT-3′SEQ ID NO.8

(2) Fluorescence quantitative PCR reaction system is as shown in Table 5,

table 5

(3) PCR reaction program

The PCR reaction program is as follows: 95°C for 10 min; 95°C for 15 sec, 60°C for 1 min, a total of 40 cycles. After the reaction program is set, fill in the melting curve and the reaction starts. After the reaction, copy out the data, and use the 2 ^-ΔΔCT method to process the obtained data. Each gene experiment was repeated 3 times, and 3 parallel samples were made each time.

Example 3

Sequence Homology and Homology Analysis of Maize ZmC3H54 Gene

As shown in Figure 1, it is a comparison diagram of the amino acid sequence homology of ZmC3H54 of the present invention and the conserved region of CCCH family zinc finger proteins of other species; according to the sequence sequencing results, the sequence comparison is carried out in the NCBI database, and the cloned gene is found The sequence has the closest homologous relationship with the CCCH zinc finger protein family. Comparing this transcription factor with the known protein sequences of other CCCH-type zinc finger protein family members, it is analyzed that its zinc finger structure type is CCCH type, and according to the structural characteristics of the DNA binding domain, the structure of the ZmC3H54 protein is CX ₈ CX ₅ CX ₃ H.

As shown in Figure 2, it is the phylogenetic tree after the amino acid sequence alignment of ZmC3H54 of the present invention and the CCCH zinc finger protein family with reported functions in Arabidopsis and other plants; in order to further analyze ZmC3H54 and other CCCH zinc finger proteins with reported functions The phylogenetic relationship of finger proteins was analyzed by analyzing the phylogenetic relationship between CCCH-type zinc finger proteins and ZmC3H54 in different plants. Through homologous sequence comparison and cluster analysis, it is found that ZmC3H54 is very close in phylogenetic relationship to a small number of zinc finger proteins, and they

Constitute a unique branch in the entire phylogenetic tree, that is, become a unique subfamily. The structure of members in this subfamily is relatively conservative, and most of the members that have been reported are related to adversity stress, and ZmC3H54 is in a branch. AtC3H47 (AtSZF1), AtC3H29(AtSZF1) and CaKR1 have been reported to be involved in abiotic stress in plants.

Example 4

The acquisition of genetically modified rice

The plant expression vector p1301a-ZmC3H54 constructed in Example 1 was introduced into the rice variety Zhonghua through the Agrobacterium-mediated rice genetic transformation method, and it was hygromycin resistant after pre-cultivation, dipping, co-cultivation, and screening callus, differentiation, rooting, seedling hardening, transplanting, and obtained 25 seedling plants. In order to screen positive transgenic seedlings, the leaves of the seedlings were cut into 0.5-1 cm lengths, immersed in Gus staining solution, stained in a 37°C incubator for 12 hours, decolorized with 75% ethanol to remove chlorophyll, and then observed, and finally 16 Transgenic rice seedlings.

Example 5

Obtaining transgenic Arabidopsis

(1) Transformation of Arabidopsis thaliana by inflorescence infiltration

Shake bacteria: Take 100 μl of the Agrobacterium broth of the overexpression vector in Example 1, add it to 100ml LB or YEP liquid medium, add the corresponding resistance, and culture overnight at 28°C.

1. Buffer configuration:

Use NaOH to adjust the pH value to 5.8, add surfactant: add 300 microliters per liter of Buffer

2. Prepare transformation solution:

Centrifuge the bacterial solution shaken overnight at 28°C to collect the bacterial cells, use a 50ml centrifuge tube, centrifuge at 4000r/min for 10min, add 25ml Buffer, and blow the precipitate to resuspend.

3. Conversion

Select six pots of Arabidopsis thaliana in the flowering stage, about 25 plants, and drop the transformation liquid prepared in the previous step on the unopened flowers and apical meristems of Arabidopsis thaliana with a straw, and pay attention to let the bacterial liquid hang on the flowers as much as possible to improve the transformation efficiency. In order to ensure the transformation efficiency, the bacterial solution was added dropwise twice. After the transformation, wrap all the plants with plastic wrap to prevent the bacterial liquid from drying up too quickly, which will affect the transformation efficiency. The plastic wrap can be removed after 24 hours. Generally, 1-2 conversions are required, and the second conversion is selected four to five days after the first conversion.

(2) Screening of Arabidopsis positive seedlings

1. Preliminary screening of seeds, planting the received seeds on a medium containing screening agents such as kanamycin/hygromycin, etc., and screening out 35 resistant seedlings.

2. Take part of the leaves and perform Gus staining on the screened seedlings, and finally obtain 20 transgenic Arabidopsis plants.

Example 6

Identification of Drought Tolerance of ZmC3H54 Transgenic Rice Plants

The plant expression vector in Example 1 was transformed into Agrobacterium EHA105, and then the rice callus was infected, and after co-cultivation and antibiotic selection, transgenic plants overexpressing the ZmC3H54 gene were obtained.

Take the rice seeds of ZmC3H54 gene rice strain L4 and L6 as the experimental group and the control strain (Zhonghua rice strain) and place them in a petri dish, soak them in deionized water to make them imbibition and germinate, and when the seedlings grow to about 10cm In the experimental group, the seedlings that can be dyed by GUS and have the same growth were transferred together with the control group to a square pot with nutrient soil: vermiculite = 1: 1, planted in equal amounts in different areas, and each line was set up with three replicates. After four weeks of cultivation in the solar greenhouse, drought treatment was carried out. After four weeks of cultivation, the transgenic rice and control rice seedlings were poured with saturated water, and this time was regarded as the 0th day of treatment. After 30 days of drought stress, the culture was resumed for 20 days, and the phenotype was observed and the survival rate was counted. As shown in Fig. 4, the transgenic ZmC3H54 rice of the present invention improves the tolerance of plants to drought.

Example 7

Sensitivity Analysis of ZmC3H54 Transgenic Rice Plants to ABA

As shown in Figure 5, the transgenic ZmC3H54 rice of the present invention has improved the sensitivity of the plant to ABA; A-C: the growth phenotype, seedling height and root length of wild-type and transgenic seedlings in the MS nutrient solution of different concentrations of ABA ;

Seeds of T2 transgenic ZmC3H54 gene L4, L6 and L8 rice lines were sown on 1/2 MS medium containing different concentrations of ABA 0 μM, 1 μM, 3 μM, and 5 μM. Under 16h light/8h darkness to make it germinate and grow. After 2 weeks of cultivation, the growth phenotype of the seedlings was observed and compared, including the height and root length of the seedlings. The experiment was repeated 3 times.

It can be clearly seen from the figure that in the culture solution containing ABA, the growth of wild-type and transgenic rice seedlings was inhibited, while the degree of inhibition of transgenic seedlings was significantly greater than that of wild-type, mainly manifested in the average stem height and root length of transgenic seedlings. Shorter than the control group, with the increase of ABA concentration 5μM, the growth inhibition of transgenic seedlings was more obvious, and the transgenic seedlings almost did not grow roots. These results indicated that overexpression of ZmC3H54 could significantly increase the sensitivity of transgenic plants to ABA.

Example 8

Sensitivity Analysis of ZmC3H54 Transgenic Arabidopsis Plants to ABA

As shown in Figure 7, it is the comparison chart of improving the ABA sensitivity of transgenic ZmC3H54 Arabidopsis thaliana of the present invention; A: phenotype on MS medium; B: phenotype on MS+0.2ABA medium; C MS Phenotype on +0.4ABA medium; D: germination rate on three plates;

Take the T2 generation seeds of Arabidopsis thaliana L20, L29, L35 strains transfected with the ZmC3H54 gene and the T2 generation seeds of the transgenic plants transformed with the pCAMBIA1301a empty vector, disinfect them with 12% bleach for 15 minutes, and then wash them with sterilized water for 5-6 times , after disinfection, place at 4°C for vernalization for 3 days. After spring flowering, put them into MS medium containing different concentrations of ABA, 0μM, 0.2μM, and 0.4μM, and place them in the Arabidopsis cultivation greenhouse after sealing. It germinates and grows, and its germination number and phenotypic changes are observed during the period. The experiment was repeated three times. It can be seen from the figure that compared with the control group, the ZmC3H54 transgenic Arabidopsis lines increased their sensitivity to ABA.

From Example 7 and Example 8, it can be further speculated that the ZmC3H54 gene may be involved in the ABA-mediated signaling pathway, and play a certain role in the germination of seeds and seedling development.

Example 9

Expression of ZmC3H54 gene in transgenic Arabidopsis

A method of the present invention for in vitro detection of ZmC3H54 gene expression in plants after induction and expression differences in various tissues in plants, comprising the following steps:

Get transgenic Arabidopsis thaliana leaf treatment samples and several different plant tissue samples of each time period after corresponding induction; transformants, respectively L20, L29, L35, L9;

(2) Extract the RNA of each sample with the Trizol method;

(3) DNA was digested with DNaseI, Arabidopsis RNA was extracted and then reverse-transcribed into cDNA, which was used as a template;

(4) Detection of the purity and content of total RNA: Take 1 μL of total RNA sample and add it to the micro-spectrophotometer probe to detect the purity and content of total RNA;

The expression of the target gene ZmC3H54 was analyzed by fluorescence quantitative and semi-quantitative PCR methods, and wild-type rice was detected as a negative control.

The detection primer sequence of fluorescence quantitative PCR, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.3, and the nucleotide sequence of described downstream primer is as sequence Shown in the list SEQ ID NO.4.

Using the Arabidopsis Actin gene as an endogenous reference, an appropriate semi-quantitative PCR reaction system was established by adjusting parameters such as annealing temperature, cycle times, and template amount.

The nucleotide sequence of the Arabidopsis internal reference gene Actin primer is

qPCR-AtActin-F 5′-TCGTTGCCCCTCCAGAGA-3′SEQ ID NO.9

qPCR-AtActin-R 5′-TACTCTGCCTTTGCGATCCA-3′SEQ ID NO.10

As shown in Figure 6, Figure 6 is the expression analysis of ZmC3H54 of the present invention in wild-type (WT) Arabidopsis and T1 generation transgenic plants; A: fluorescence quantitative analysis; B: semi-quantitative analysis; ZmC3H54 can be expressed in transgenic lines normal expression in different strains, and the expression levels in different strains are quite different, among which the expression levels of the selected L29 and L35 strains are relatively high, and the expression levels of other transgenic lines are relatively low, which may be related to the target gene fragment. The insertion site is related to the copy number. However, the expression of the target gene was not detected in wild-type rice.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

Claims (10)

1. A corn CCCH type zinc finger protein, its amino acid sequence is as shown in the sequence table SEQ ID NO.1. 2. the coding gene ZmC3H54 of the corn CCCH type zinc finger protein described in claim 1, its nucleotide sequence is as shown in sequence table SEQ ID NO.2. 3. A recombinant vector, a recombinant bacterium, or a transgenic cell line containing the coding gene ZmC3H54 described in claim 2. 4. The application of the maize CCCH-type zinc finger protein according to claim 1 or the coding gene ZmC3H54 of the maize CCCH-type zinc finger protein according to claim 2 in breeding transgenic plants resistant to abiotic stress. 5. The application according to claim 4, the transgenic plant is rice or Arabidopsis. 6. A method for preparing a transgenic plant resistant to abiotic stress according to claim 4, characterized in that it comprises the steps of: (1) Obtain the nucleotide sequence and amino acid sequence of the coding gene ZmC3H54 of the corn CCCH-type zinc finger protein; (2) obtain the corn ZmC3H54 gene fragment by PCR; The expression profile of maize gene ZmC3H54 under adversity stress was analyzed by fluorescent quantitative PCR method, and the ZmC3H54 gene fragment was constructed into a plasmid vector; (3) transforming the plasmid with ZmC3H54 obtained in step (2) into Agrobacterium; transforming the target plant with the Agrobacterium with the transformed plasmid; (4) Screening and identification of transgenic positive seedlings of target plants; analysis of abiotic stress resistance of transgenic homozygous plants. 7. the preparation method of the transgenic plant of anti-abiotic adversity stress according to claim 6 is characterized in that: in step (1), after the cloning vector plasmid that will contain ZmC3H54 gene is through BamHI and XbaI double-digestion, utilize The DNA recovery kit recovers the 1119bp DNA fragment of SEQ ID NO.2, connects this fragment with the corresponding enzyme-cut pCAMBIA1301a vector, and the obtained vector is named p1301a-ZmC3H54 recombinant vector; The primer sequence of the gene is cloned from corn cDNA as a template PCR, and the primer of the amplified ZmC3H54 gene is divided into an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in the sequence table SEQ ID NO.5 , the nucleotide sequence of the upstream primer is shown in the sequence table SEQ ID NO.6; In step (3), the target plant is rice or Arabidopsis. In step (4), the abiotic stress resistance analysis includes drought tolerance analysis and sensitivity analysis of plant seed germination to plant hormone ABA. 8. A method for in vitro detection of ZmC3H54 gene expression in plants after induction and expression differences in various tissues in plants, characterized in that it comprises the following steps: (1) Take the leaf treatment samples and several different tissue samples of the plants at various time periods after the corresponding induction of the plants; (2) Extract the RNA of each sample with the Trizol method; (3) Digest the DNA with DnaseI, then reverse transcribe to form cDNA, and use it as a template; (4) Analyze with fluorescent quantitative PCR, the detection primer sequence of fluorescent quantitative PCR, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of the upstream primer is shown in the sequence listing SEQ ID NO.3, and the nucleotide sequence of the upstream primer is shown in the sequence listing SEQ ID NO.4. 9. according to claim 8, be used for in vitro detection ZmC3H54 gene expression level in plant after induction and the method for expression level difference in each tissue in plant, it is characterized in that: in step (1), described plant is corn; Stress treatment sampling: the plant material is corn B73 inbred line. When the corn seedlings grow to the three-leaf stage, the normal irrigated plants are used as the control, and the stress treatments of 10% PEG solution, 100uM ABA solution and 200mmol/L NaCl solution are respectively carried out , samples were taken after treatment for different time periods, the time was 0h, 1h, 3h, 6h, 12h, 24h respectively; the leaves of the same part of the corn seedlings of the test group and the control group were cut with scissors, and three samples were taken at each treatment stage; , the samples were stored frozen in liquid nitrogen at -80°C; Tissue sampling: In order to analyze the difference in the expression level of the maize ZmC3H54 gene in various tissues, 8 representative tissues of maize were selected, and the representative tissues were root, stem, young leaf, silk, tassel, pre-pollination Ears, bracts, embryos and endosperms were sampled and stored in liquid nitrogen at -80°C; In step (4), the detection of total RNA purity and content: take 1 μ L of total RNA sample and add it to the micro-spectrophotometer probe to detect the total RNA purity and content; Get normal corn as contrast, corn internal reference gene Actin primer, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as shown in sequence table SEQ ID NO.7, the nucleotide sequence of described upstream primer The nucleotide sequence is shown in the sequence listing SEQ ID NO.8. 10. according to claim 8, be used for in vitro detection ZmC3H54 gene in the method for the expression amount in induced plant and the expression amount difference in each tissue in plant, it is characterized in that: In step (4), get normal Arabidopsis thaliana as contrast, Arabidopsis internal reference gene Actin primer, described primer is divided into upstream primer and downstream primer; The nucleotide sequence of described upstream primer is as sequence table SEQ ID As shown in NO.9, the nucleotide sequence of the upstream primer is shown in the sequence table SEQ ID NO.10.