CN112812161A

CN112812161A - Application of protein IbMYC2 in regulation and control of plant drought resistance

Info

Publication number: CN112812161A
Application number: CN202110053146.2A
Authority: CN
Inventors: 翟红; 刘庆昌; 何绍贞; 赵宁; 赵宏媛
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2021-05-18
Anticipated expiration: 2041-01-15
Also published as: CN112812161B

Abstract

The invention discloses application of protein IbMYC2 in regulation and control of plant drought resistance. The amino acid sequence of the protein IbMYC2 provided by the invention is shown in SEQ IDNO: 2. Experiments prove that the IbMYC2 gene is overexpressed in Arabidopsis, so that the drought resistance of Arabidopsis can be improved, and the improvement of the drought resistance is shown as follows: increased survival rate, increased root length, increased fresh weight, increased JA content, increased SOD enzyme activity, decreased MDA content and H under drought stress₂O₂The content is reduced. Therefore, the protein IbMYC2 can regulate the drought resistance of plants. The invention has important application value.

Description

Application of protein IbMYC2 in regulation and control of plant drought resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a protein IbMYC2 in regulation and control of plant drought resistance.

Background

Drought is the most important environmental factor affecting plant growth and crop yield. Drought becomes a serious problem affecting agricultural production, and the key problem and the important problem which need to be solved urgently in new variety cultivation are that the drought resistance of crops is improved by using a genetic engineering means, and the adaptability of the crops and the economic crops to the adverse environment is improved. In recent years, a great deal of research is carried out on the mechanism of plants responding to adverse environmental stress such as drought and the like from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, abundant data are accumulated, particularly with the development of molecular biology, people can know the adverse resistance mechanism of the plants to the drought stress on the molecular level of gene composition, expression regulation, signal conduction and the like, and a new way is developed for improving the stress resistance of the plants by utilizing a gene engineering means. Due to the complexity of the stress resistance of plants, the traditional breeding method is very difficult to improve the stress resistance of the plants, with the development of molecular biology, a new approach for the stress resistance breeding of the plants is developed by a genetic engineering means, but the separation of high-efficiency stress resistance genes becomes a main factor for limiting the stress resistance genetic engineering of the plants.

Disclosure of Invention

The invention aims to improve the drought resistance of plants.

The invention firstly protects the IbMYC2 protein derived from sweet potatoes, which can be 1) or 2) or 3) or 4):

1) the amino acid sequence is protein shown as SEQ ID NO. 2;

2) 2, the N end or/and the C end of the protein shown in SEQ ID NO.2 is connected with a label to obtain fusion protein;

3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in 1) or 2), is derived from sweet potatoes and is related to drought resistance;

4) protein which has 80 percent or more than 80 percent of homology with the amino acid sequence limited by SEQ ID NO.2, is derived from sweet potatoes and is related to drought resistance.

Wherein, SEQ ID NO 2 consists of 472 amino acid residues.

In order to facilitate the purification of the protein of 1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxyl terminus of the protein shown in SEQ ID NO: 2.

TABLE 1 sequence of tags

The protein according to 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in 3) above can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of 3) above can be obtained by deleting one or several amino acid residues of the codon in the DNA sequence shown in SEQ ID NO.1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

The invention also protects a nucleic acid molecule for coding the protein IbMYC 2.

The nucleic acid molecule encoding the protein IbMYC2 can be a DNA molecule shown in (a1), or (a2), or (a3), or (a 4):

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

(a2) DNA molecule with the nucleotide sequence shown as SEQ ID NO. 1;

(a3) a DNA molecule which has 75 percent or more homology with the nucleotide sequence limited by (a1) or (a2), is derived from sweet potato and codes any one of the proteins IbMYC 2;

(a4) a DNA molecule which is derived from sweet potato and encodes any one of the proteins IbMYC2 and is hybridized with the nucleotide sequence defined in (a1) or (a2) under strict conditions.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO.1 consists of 1419 nucleotides, and the nucleotide of SEQ ID NO.1 encodes an amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence of the invention encoding the protein IbMYC2 can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of the protein IbMYC2 isolated by the invention as long as the nucleotide sequence encodes the protein IbMYC2, are derived from and identical to the nucleotide sequence of the invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that is 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of the present invention encoding the protein IbMYC2 consisting of the amino acid sequence set forth in SEQ ID NO. 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The invention also protects an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules.

The recombinant vector containing any one of the nucleic acid molecules can be obtained by inserting the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing.

The recombinant vector can be specifically a recombinant plasmid pCB-IbMYC 2. The recombinant plasmid pCB-IbMYC2 can be a recombinant plasmid obtained by replacing a small fragment between recognition sequences of restriction enzymes KpnI and SalI of a vector pCAMBIA super1300-GFP with a DNA molecule shown in SEQ ID NO. 1.

The recombinant microorganism containing any of the above-described nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-described nucleic acid molecules into a starting microorganism.

The starting microorganism can be agrobacterium or escherichia coli. The agrobacterium may specifically be agrobacterium tumefaciens. The agrobacterium tumefaciens can be specifically agrobacterium tumefaciens GV 3101.

The recombinant microorganism containing any one of the above nucleic acid molecules can be GV3101/pCB-IbMYC 2. The GV3101/pCB-IbMYC2 can be a recombinant agrobacterium obtained by introducing a recombinant plasmid pCB-IbMYC2 into Agrobacterium tumefaciens GV 3101.

The invention also provides application of any one of the protein IbMYC2, any one of the nucleic acid molecules or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules in regulation and control of plant drought resistance.

The invention also provides application of any one of the protein IbMYC2, any one of the nucleic acid molecules or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing any one of the nucleic acid molecules in cultivation of a transgenic plant with changed drought resistance.

In any of the above applications, the regulating drought resistance of a plant may be to improve drought resistance of a plant.

In any of the above applications, the cultivation of a transgenic plant with altered drought resistance may be a cultivation of a transgenic plant with improved drought resistance.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: increasing the expression level and/or activity of the protein IbMYC2 in a receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance as compared to the recipient plant.

In the method, the effect of improving the expression level and/or activity of any one of the proteins IbMYC2 in the recipient plant can be achieved by a method well known in the art, such as transgenosis, multi-copy, promoter change, regulatory factor change and the like.

In the above method, the "increasing the expression level and/or activity of any one of the proteins IbMYC2 in a recipient plant" may be specifically achieved by introducing a nucleic acid molecule encoding the protein IbMYC2 into the recipient plant.

In the above method, the nucleic acid molecule encoding the protein IbMYC2 may be a DNA molecule represented by (a1) or (a2) or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

(a2) DNA molecule with the nucleotide sequence shown as SEQ ID NO. 1;

The introduction of a nucleic acid molecule encoding the protein IbMYC2 into a recipient plant may be specifically achieved by introducing a recombinant vector comprising any of the nucleic acid molecules described above into a recipient plant.

The recombinant vector containing any one of the nucleic acid molecules can be specifically a recombinant plasmid pCB-IbMYC 2. The recombinant plasmid pCB-IbMYC2 can be a recombinant plasmid obtained by replacing a small fragment between recognition sequences of restriction enzymes KpnI and SalI of a vector pCAMBIA super1300-GFP with a DNA molecule shown in SEQ ID NO. 1.

The invention also provides a plant breeding method, which comprises the following steps: increasing the expression level and/or activity of the protein IbMYC2 in the plant, thereby improving the drought resistance of the plant.

Any of the plants described above may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) wild type Arabidopsis thaliana Col-0.

The above-mentioned improvement in drought resistance can be manifested by an increase in the survival rate under drought stress, an increase in the root length, an increase in the fresh weight, an increase in the JA content, an increase in the SOD enzyme activity, a decrease in the MDA content and H₂O₂At least one of reduced content.

Experiments prove that the IbMYC2 gene is overexpressed in Arabidopsis, so that the drought resistance of Arabidopsis can be improved, and the improvement of the drought resistance is shown as follows: increased survival rate, increased root length, increased fresh weight, increased JA content, increased SOD enzyme activity, decreased MDA content and H under drought stress₂O₂The content is reduced. Therefore, the protein IbMYC2 can regulate the drought resistance of plants. The invention has important application value.

Drawings

FIG. 1 shows 8T₃The molecular identification result of the generation homozygous IbMYC2 transgenic Arabidopsis thaliana.

FIG. 2 shows the real-time quantitative PCR detection of 8T₃The expression level of the IbMYC2 gene of the transgenic Arabidopsis IbMYC2 gene is homozygous.

FIG. 3 shows the drought resistance test result in step three 1 of example 2.

FIG. 4 shows the drought resistance test results obtained in step three 2 of example 2.

FIG. 5 shows the results of measurement of physiological and biochemical parameters in step three 3 of example 2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Wild type Arabidopsis thaliana Col-0 is described in the following documents: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004,36: 167-. The wild type Arabidopsis thaliana Col-0 is hereinafter abbreviated as wild type Arabidopsis thaliana or WT.

Sweet potato strain JS6-5 is described in the following documents: ZHao HY, ZHang SS, Wang FB, ZHao N, He SZ, Liu QC, ZHai H.comprehensive transfer analysis of pure-fly sweet potato products identifications into the molecular mechanism of anticancer in biosyntheses. frontiers of Agricultural Science and Engineering, 2018, doi.org/10.15302/J-FASE-2018219. The public is available from sweet potato genetic breeding research laboratory of Chinese agriculture university to repeat the experiment.

The plant total RNA extraction kit is a Transzol Up plant total RNA extraction kit (full-scale gold, catalog number ET 111). The pEASY-Blunt simple vector is a product of Beijing Quanyujin Biotechnology Co., Ltd. QuantScript RT Kit Quant cDNA Kit is a product of Tiangen Biochemical technology (Beijing) Co., Ltd., and has a product catalog number KR 103. The vector pCAMBIA super1300-GFP is a product of Wuhan vast Ling Biotech Co., Ltd, and the catalog number is L3080.

1/2 Hoagland nutrient solutions are described in the following documents: LiudeGao, acquisition of sweet potato plants over-expressing IbP5CR, IbERD3, IbELT and IbNFU1 genes and identification of salt tolerance.

The parameters of the light-dark alternate culture in the following examples are: the illumination time is 16h, and the dark time is 8 h.

In the following examples, the intensity of light was 3100 to 3500 Lux.

Example 1 obtaining of IbMYC2 Gene

The IbMYC2 gene is obtained by the following steps:

1. extracting the total RNA of the young leaf of the sweet potato strain JS6-5 by using a plant total RNA extraction Kit, and carrying out reverse transcription on the total RNA by using a QuantScript RT Kit Quant cDNA Kit to obtain a first strand cDNA.

2. Taking the cDNA obtained in the step 1 as a template, and adopting a primer MYC 2-F: 5'-ATGGAAGAGATTCTTTCTTCATCTT-3' and primer MYC 2-R: 5'-TTAAGACTGCAATCTTCTAAGGATG-3', obtaining a 1419bp PCR amplification product and sequencing.

The result shows that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 1. The gene shown in SEQ ID NO.1 is named as IbMYC2 gene, the coded protein is named as IbMYC2 protein or protein IbMYC2, and the amino acid sequence is shown in SEQ ID NO. 2.

Example 2 application of IbMYC2 protein in controlling drought resistance of arabidopsis thaliana

Construction of recombinant plasmid

1. The vector pCAMBIA super1300-GFP was digested with restriction enzymes KpnI and SalI, and the 10783bp vector backbone was recovered.

2. Artificially synthesizing a double-stranded DNA molecule shown in SEQ ID NO. 1. Taking the double-stranded DNA molecule as a template, and taking a primer MYC 2-OE-F: 5' -GGGGTACCATGGAAGAGATTCTTTCTTCATCTT-3' (recognition sequence for restriction enzyme KpnI is underlined) and primer MYC 2-OE-R: 5' -ACGCGTCGACAGACTGCAATCTTCTAAGGATGAC-3' (the recognition sequence of the restriction enzyme SalI is underlined) was subjected to PCR amplification to obtain a double-stranded DNA molecule containing the recognition sequence of the restriction enzyme.

3. And (3) connecting the double-stranded DNA molecule containing the recognition sequence of the restriction enzyme obtained in the step (2) to a pEASY-Blunt simple vector to obtain an intermediate vector.

4. The intermediate vector was double-digested with restriction enzymes KpnI and SalI, and a DNA fragment of about 1400bp was recovered.

5. And connecting the DNA fragment with a vector framework to obtain a recombinant plasmid pCB-IbMYC 2.

The recombinant plasmid pCB-IbMYC2 was sequenced. According to the sequencing result, the structure of the recombinant plasmid pCB-IbMYC2 is described as follows: the small fragment between the recognition sequences of restriction enzymes KpnI and SalI of the vector pCAMBIA super1300-GFP was replaced with the DNA molecule shown in SEQ ID NO.1 to obtain a recombinant plasmid. The recombinant plasmid pCB-IbMYC2 expresses IbMYC2 protein shown in SEQ ID NO. 2.

Second, obtaining of IbMYC2 transgenic Arabidopsis

1. The recombinant plasmid pCB-IbMYC2 is used for transforming agrobacterium tumefaciens GV3101 to obtain recombinant agrobacterium tumefaciens which is named as GV3101/pCB-IbMYC 2.

2. GV3101/pCB-IbMYC2 was transferred to wild type Arabidopsis thaliana by the Arabidopsis thaliana inflorescence floral dip transformation method (Clough, S.J., andBi, A.F.. Floraldip: asipli and ethod for Agrobacterium-meditedtransformation of Arabidopsis thaliana.plant J. (1998)16, 735-₁The seed of Arabidopsis thaliana with IbMYC2 gene transfer is simulated.

3. Will T₁Planting seeds of Arabidopsis thaliana with IbMYC2 gene transfer on 1/2MS solid culture medium containing 12.5mg/LPPT, purifying at 4 deg.C for 3 days, culturing at 22 deg.C for 7-10 days, and obtaining Arabidopsis thaliana (resistant seedling) capable of normally growing as T₁The IbMYC2 gene positive seedling is transferred. T is₁The seeds received by the positive seedlings of the IbMYC2 gene are T₂Seeds of Arabidopsis thaliana with IbMYC2 gene.

4. The T of different strains screened out in the step 3₂Seeds of Arabidopsis thaliana transformed with IbMYC2 gene are sown on 1/2MS solid medium containing 12.5mg/LPPT for screening, if the ratio of the number of Arabidopsis thaliana (resistant seedlings) capable of normally growing to the number of Arabidopsis thaliana (non-resistant seedlings) incapable of normally growing in a certain strain is 3: 1, the strain is a strain with an IbMYC2 gene inserted into one copy, and the seeds received by the resistant seedlings in the strain are T₃Seeds of Arabidopsis thaliana with IbMYC2 gene.

5. The T screened out in the step 4₃Seeds of arabidopsis thaliana transformed with IbMYC2 gene are sown on 1/2MS solid culture medium containing 12.5mg/LPPT again for screening, and the seeds which are all resistant seedlings are T₃The transgenic IbMYC2 transgenic Arabidopsis thaliana is homozygous for the generation.

50T to be screened₃The generation homozygous IbMYC2 transgenic Arabidopsis strains are sequentially named as L1-L50.

6. Molecular identification

The Arabidopsis thaliana to be tested is wild type Arabidopsis thaliana and L1-L50.

Each Arabidopsis to be tested is subjected to the following steps:

(1) and (3) sowing 5 seeds of arabidopsis thaliana to be detected on 1/2MS solid culture medium, purifying for 3 days at 4 ℃, and then alternately culturing for 8 days in light and dark at 22 ℃ to obtain arabidopsis thaliana seedlings to be detected.

(2) After the step (1) is completed, extracting genome DNA of the leaves of the arabidopsis seedlings to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of a primer MYC2-F and a primer MYC2-R to obtain a PCR amplification product; performing PCR amplification by using water as a template and a primer pair consisting of a primer MYC2-F and a primer MYC2-R to obtain a PCR amplification product as a negative control; the recombinant plasmid pCB-IbMYC2 is used as a template, and a primer pair consisting of a primer MYC2-F and a primer MYC2-R is used for PCR amplification to obtain a PCR amplification product which is used as a positive control.

(3) After completion of step (2), each PCR amplification product was subjected to 1% (w/v) agarose gel electrophoresis, followed by judgment as follows: if with a certain T₃The PCR amplification product obtained by using the genome DNA of the generation homozygous IbMYC2 gene Arabidopsis thaliana as a template contains a DNA fragment (the same as the positive control fragment) of about 1419bp, so that the T is₃The generation homozygous IbMYC2 transgenic Arabidopsis is a positive plant; otherwise, the plant is not a positive plant.

Part of the detection results are shown in FIG. 1(M is DNA Marker, W is negative control, P is positive control, WT is wild type Arabidopsis thaliana). The results show that the PCR amplification products of L1, L2, L3, L20, L25, L30, L43, L44 and the positive control all contain a DNA fragment of 1419bp, and the PCR amplification products of the negative control and the wild type Arabidopsis do not contain a DNA fragment of 1419 bp. It can be seen that L1, L2, L3, L20, L25, L30, L43 and L44 are all positive plants.

7. Real-time quantitative PCR detection of T₃Expression level of generation homozygous transgenic IbMYC2 gene Arabidopsis IbMYC2 gene

The arabidopsis seeds to be detected are wild arabidopsis seeds, seeds of L1, seeds of L2, seeds of L3, seeds of L20, seeds of L25, seeds of L30, seeds of L43 or seeds of L44.

(1) And (3) sowing 5 arabidopsis thaliana seeds to be detected on 1/2MS solid culture medium, purifying for 3 days at 4 ℃, and then alternately culturing for 8 days in light and dark at 22 ℃ to obtain arabidopsis thaliana seedlings to be detected.

(2) After the step (1) is finished, extracting the total RNA of each arabidopsis seedling to be detected, and reversing by using reverse transcriptase to obtain cDNA; the relative expression level of the IbMYC2 gene in the cDNA was detected by real-time quantitative PCR (with Arabidopsis actin gene as reference gene).

The primer for detecting the IbMYC2 gene is qMYC 2-F: 5'-CCATACATGAGCACTGGGAATTG-3' and qMYC 2-R: 5'-AGGGGAAAGACATTGGTTAAGATCT-3' are provided.

The primers for detecting the actin gene are actin-F: 5'-GCACCCTGTTCTTCTTACCGA-3' and actin-R: 5'-AGTAAGGTCACGTCCAGCAAGG-3' are provided.

The results are shown in FIG. 2. The result shows that the IbMYC2 gene is an arabidopsis thaliana foreign gene and is hardly expressed in wild arabidopsis thaliana, but the IbMYC2 gene is 8T genes₃The strains of the transgenic IbMYC2 Arabidopsis thaliana (L1, L2, L3, L20, L25, L30, L43 and L44 respectively) have different expression degrees.

Three strains (namely L1, L3 and L20) with the highest expression level of the IbMYC2 gene are selected for subsequent experiments.

Thirdly, drought resistance identification of IbMYC2 transgenic Arabidopsis thaliana

1. Identification of drought resistance of IbMYC2 transgenic Arabidopsis

The Arabidopsis seeds to be detected are wild Arabidopsis seeds, L1 seeds, L3 seeds or L20 seeds.

(1) Taking the arabidopsis seeds to be tested, sterilizing the arabidopsis seeds for 10min by using a 2.6% (v/v) sodium hypochlorite aqueous solution, and then washing the arabidopsis seeds for three times by using sterilized water.

(2) And (3) after the step (1) is finished, sowing the arabidopsis thaliana seeds in 1/2MS solid culture medium, purifying for 3 days at 4 ℃, and then alternately culturing in light and dark at 22 ℃ until cotyledons are completely expanded to obtain the arabidopsis thaliana seedlings to be detected.

(3) After the step (2) is completed, the Arabidopsis seedlings to be tested with basically consistent growth vigor are transferred to 1/2MS solid medium or 1/2MS solid medium containing 350mM mannitol, and are cultured alternately in dark and light at 22 ℃ (upright culture) for 1 week, and the growth state of Arabidopsis is observed.

The growth state of Arabidopsis thaliana is shown in the left panel of FIG. 3.

(4) And (4) after the step (3) is completed, counting the root length and the fresh weight of the arabidopsis seedlings (the experiment is repeated three times, and the average value is taken, wherein the counted arabidopsis seedlings are 4 plants each time).

The detection results are shown in the right panel of FIG. 3.

The results showed that wild type Arabidopsis thaliana and T were cultured on 1/2MS solid medium₃The generation homozygous IbMYC2 transgenic Arabidopsis (namely L1, L3 and L20) has no significant difference in root length and fresh weight; t in 1/2MS solid Medium containing 350mM mannitol, in comparison with wild type Arabidopsis thaliana₃The root length and the fresh weight of the generation-homozygous IbMYC2 transgenic Arabidopsis (namely L1, L3 and L20) are both increased obviously.

2. Identification of drought resistance of IbMYC2 transgenic Arabidopsis thaliana

(2) After the step (1) is finished, sowing arabidopsis thaliana seeds in 1/2MS solid culture medium, purifying for 3 days at 4 ℃, and alternately culturing for 7 days in light and dark at 22 ℃ to obtain arabidopsis thaliana seedlings to be detected; and randomly transplanting the arabidopsis seedlings to be detected with basically consistent growth states to flowerpots filled with nutrient soil, and normally culturing for 10 days to obtain the arabidopsis seedlings to be detected before treatment.

In order to ensure that the experimental conditions are consistent as much as possible, the weight of the nutrient soil in each flowerpot is the same, and the number of the arabidopsis seedlings to be tested transplanted in each flowerpot is also the same.

(3) After the step (2) is completed, taking the arabidopsis seedlings to be detected before treatment, and performing the following treatment:

control (1 pot): culturing for 3 weeks; irrigating 50mL of 1/2 Hoagland nutrient solution every 2 days;

drought stress (1 pot): culturing for 3 weeks; no water was poured during the cultivation.

Rehydration was carried out for 2 days after drought stress.

(4) And (4) after the step (3) is finished, observing the growth state of the arabidopsis seedlings to be detected after treatment.

The growth state of the Arabidopsis seedlings to be tested after the treatment is shown in figure 4.

The results show that, under normal culture conditions (i.e., control), wild-type Arabidopsis thaliana and T₃The generation homozygous IbMYC2 transgenic Arabidopsis (namely L1, L3 and L20) all grow vigorously; under the condition of drought stress, the wild arabidopsis thaliana can not be recovered after all withering death and rehydration, and T₃The generation homozygous IbMYC2 transgenic Arabidopsis (namely L1, L3 and L20) has yellow leaves, and the leaves can absorb water and stretch back to green after rehydration, so that the growth state of the plant is good.

3. Measurement of physiological and biochemical indexes

(1) The JA content is detected by taking the aerial parts of the Arabidopsis seedlings to be detected which are treated for 2 weeks in the step 2 (3) by contrast or the aerial parts of the Arabidopsis seedlings to be detected which are treated for 2 weeks by drought stress according to the method described in the literature (Yan Li, Huan Zhang, Qian Zhang, Qingchang Liu, Hong Zhai, Ning Zhao, Shaozhen He, An AP2/ERF gene, IbRAP2-12, from sweet potato in fermented in saline and moist in transgenic Arabidopsis plant Science, 2019, 281: 19-30).

(2) Taking the arabidopsis seedlings to be detected which are subjected to contrast treatment for 2 weeks in the step 2 (3) or the arabidopsis seedlings to be detected which are subjected to drought stress treatment for 2 weeks, adopting a superoxide dismutase (SOD) kit (Suzhou ke mingguan organism, catalog number SOD-1-Y) to detect SOD enzyme activity, adopting a Malondialdehyde (MDA) kit (Suzhou ke mingguan organism, catalog number MDA-2-Y) to detect MDA content, and adopting hydrogen peroxide (H) to detect₂O₂) Kit (Suzhou ke ming bio, catalog number H)₂O₂-2-Y) detection of H₂O₂And (4) content.

The above experiment was repeated three times and the average value was taken, and 10 Arabidopsis seedlings to be tested were tested each time.

The results are shown in FIG. 5. The results show that, under normal culture conditions (i.e., control), wild-type Arabidopsis thaliana and T₃JA content, SOD enzyme activity, MDA content and H of transgenic IbMYC2 arabidopsis thaliana (namely L1, L3 and L20) of generation-homozygous transgenic IbMYC2 gene₂O₂The contents have no obvious difference; t compared to wild type Arabidopsis under drought stress conditions₃The SOD enzyme activity and JA content of the generation homozygous IbMYC2 transgenic arabidopsis thaliana (namely L1, L3 and L20) are both obviously increased, and the MDA content and H content₂O₂The content is obviously reduced.

The results show that the drought resistance of arabidopsis can be obviously improved by over-expressing the IbMYC2 gene; the drought resistance is improved as follows: increased survival rate, increased root length, increased fresh weight, increased JA content, increased SOD enzyme activity, decreased MDA content, and H₂O₂The content is reduced.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> university of agriculture in China

Application of <120> protein IbMYC2 in regulation and control of plant drought resistance

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<170> PatentIn version 3.5

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aacgtgtcca agatggacaa ggcctccttg ctagccgacg cggtcaccta catcaaccgc 1020

ctcaaggcta aggttggaga tctggagaac aaactgggaa cgcacggcga gaaccagatg 1080

tccaggaagc gaatcatgga gataatgcac gacgcgcaga gcaccaccac gtccacggtg 1140

gaccacgtga tgggcggcgg atgtgcgttc ggggcgatgg acgttgaggt caagatcatc 1200

gggtcggagg ccatgatccg ggtccattct cccgacctca attatccggc ggctaggcta 1260

atgaatgtgc ttagagaaat ggagctgaag atccaccacg ccagtgtgtc cagcgtcagg 1320

gatttgatgc ttcaagatgt tgtgattagg gttccagacg ggttgaccaa tgaggaagat 1380

gccctaaaag ctgtcatcct tagaagattg cagtcttaa 1419

<210> 2

<211> 472

<212> PRT

<213> Ipomoea batatas

<400> 2

Met Glu Glu Ile Leu Ser Ser Ser Ser Ser Ser Ser Ile Pro Asn Ser

1 5 10 15

Leu Gln Lys Arg Ile Gln Phe Phe Leu His Asn Arg Pro Glu Trp Trp

20 25 30

Val Tyr Ala Ile Phe Trp Gln Ala Thr Lys Gly Gly His Gly Arg Val

35 40 45

Val Leu Ser Trp Gly Asp Gly His Phe Arg Gly Thr Lys Gly Val Gly

50 55 60

Pro Ser Arg Ser Asn Pro Leu Gln Leu Gln Asn Lys Phe Glu Gly Glu

65 70 75 80

Gly Leu Val Glu Gly Asn Val Thr Glu Tyr Ala Glu Trp Tyr Tyr Met

85 90 95

Val Ser Met Thr Lys Ser Phe Ala Ala Pro Asp Asp Leu Ile Val Gln

100 105 110

Thr Phe Asp Ser Gly Ser Tyr Ile Trp Leu Ser Asp Ser Asn Gln Val

115 120 125

Gln Phe Tyr His Ser Glu Arg Ala Lys Glu Ala His Leu His Gly Ile

130 135 140

Asn Thr Leu Val Cys Phe Ser Thr Ser Ala Gly Val Ile Glu Leu Gly

145 150 155 160

Ser Ser Asp Ser Ile His Glu His Trp Glu Leu Leu Gln Ile Gly Arg

165 170 175

Ser Ile Phe Asn Ala Gln Asn Thr Ser Leu Pro Gln Thr Gln Thr Gln

180 185 190

Thr Gln Asn Gln Asn Gln Asn Gln Ser Gln Asp Leu Phe Pro Phe Ser

195 200 205

Gln Asp Leu Asn Gln Cys Leu Ser Pro Ile Asp Phe Arg Leu Gly Asp

210 215 220

Pro His Gln Glu Lys Glu Lys Glu Ser Cys Arg Ala Val Asp Leu His

225 230 235 240

Gly Pro Asn Ser Asp Ile Lys Lys Glu Val Leu Val Ile Asn Asp Leu

245 250 255

Ser Pro Asp Ser Gly Asn Ser Asp Phe Phe Glu Thr Thr Leu Thr His

260 265 270

Gln Ser Ala Met Val Gly Ser Arg Lys Arg Ser Arg Lys Gly Ala Leu

275 280 285

Thr Gly Arg Glu Met Ala Met Asn His Val Glu Ala Glu Arg Gln Arg

290 295 300

Arg Glu Lys Leu Asn His Arg Phe Tyr Ala Leu Arg Ser Val Val Pro

305 310 315 320

Asn Val Ser Lys Met Asp Lys Ala Ser Leu Leu Ala Asp Ala Val Thr

325 330 335

Tyr Ile Asn Arg Leu Lys Ala Lys Val Gly Asp Leu Glu Asn Lys Leu

340 345 350

Gly Thr His Gly Glu Asn Gln Met Ser Arg Lys Arg Ile Met Glu Ile

355 360 365

Met His Asp Ala Gln Ser Thr Thr Thr Ser Thr Val Asp His Val Met

370 375 380

Gly Gly Gly Cys Ala Phe Gly Ala Met Asp Val Glu Val Lys Ile Ile

385 390 395 400

Gly Ser Glu Ala Met Ile Arg Val His Ser Pro Asp Leu Asn Tyr Pro

405 410 415

Ala Ala Arg Leu Met Asn Val Leu Arg Glu Met Glu Leu Lys Ile His

420 425 430

His Ala Ser Val Ser Ser Val Arg Asp Leu Met Leu Gln Asp Val Val

435 440 445

Ile Arg Val Pro Asp Gly Leu Thr Asn Glu Glu Asp Ala Leu Lys Ala

450 455 460

Val Ile Leu Arg Arg Leu Gln Ser

465 470

Claims

1. Protein IbMYC2, 1) or 2) or 3) or 4) as follows:

1) the amino acid sequence is protein shown as SEQ ID NO. 2;

2. A nucleic acid molecule encoding the protein IbMYC2 of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown in (a1), or (a2), or (a3) or (a 4):

(a1) the coding region is a DNA molecule shown in SEQ ID NO. 1;

(a2) DNA molecule with the nucleotide sequence shown as SEQ ID NO. 1;

(a3) a DNA molecule which has 75% or more homology with the nucleotide sequence defined in (a1) or (a2), is derived from sweetpotato and encodes the protein IbMYC2 of claim 1;

(a4) a DNA molecule which is derived from sweetpotato and encodes the protein IbMYC2 of claim 1, and hybridizes with the nucleotide sequence defined in (a1) or (a2) under stringent conditions.

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3.

5, b1) or b 2):

b1) use of the protein IbMYC2 of claim 1, or the nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3, for modulating drought resistance in a plant;

b2) use of the protein IbMYC2 of claim 1, or the nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3, for the production of transgenic plants with altered drought resistance.

6. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the protein IbMYC2 in the receptor plant to obtain a transgenic plant; the transgenic plant has increased drought resistance as compared to the recipient plant.

7. The method of claim 6, wherein: the improvement of the expression level and/or activity of the protein IbMYC2 in a recipient plant is realized by introducing a nucleic acid molecule encoding the protein IbMYC2 into the recipient plant.

8. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of the protein IbMYC2 in the plant according to claim 1, thereby improving the drought resistance of the plant.

9. The protein IbMYC2 of claim 1, or the use of claim 5, or the method of any one of claims 6-8, wherein: the drought resistance improvement is manifested by increased survival rate, increased root length, increased fresh weight, increased JA content, increased SOD enzyme activity, decreased MDA content and H content under drought stress₂O₂At least one of reduced content.

10. The use of claim 5, or the method of any one of claims 6 to 8, wherein: the plant is any one of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the family Dioscoreaceae; c4) sweet potato; c5) a cruciferous plant; c6) arabidopsis thaliana; c7) wild type Arabidopsis thaliana Col-0.