CN112724213A

CN112724213A - Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof

Info

Publication number: CN112724213A
Application number: CN202110042572.6A
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: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-04-30
Anticipated expiration: 2041-01-13
Also published as: CN112724213B

Abstract

The invention discloses a sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and a coding gene and application thereof. The amino acid sequence of the protein IbMYB4 provided by the invention is shown in SEQ ID NO. 2. Experiments prove that the IbMYB4 gene is overexpressed in Arabidopsis, the anthocyanin content and the stress resistance of the Arabidopsis can be improved, and the stress resistance is improved by the following steps: increased survival rate, increased root length, increased fresh weight, increased SOD enzyme activity, decreased MDA content, and H₂O₂Reduced content and increased ABA content. Therefore, the protein IbMYB4 and the coding gene thereof can regulate and control plant stress resistance and anthocyaninContent, has important application value.

Description

Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and a coding gene and application thereof.

Background

Anthocyanin is an important natural pigment that is widely distributed in flowers, fruits, seeds, leaves and storage roots of plants and gives plants red, purple and blue colors. During the growth and development of plants, the anthocyanin endows the plants with bright color so that the anthocyanin plays an important role in the process of spreading pollen and seeds by the plants. In addition, the anthocyanin plays a certain role in the aspects of biotic stress resistance and abiotic stress resistance of plants due to the characteristics of reducing ultraviolet damage, resisting diseases and insects, resisting low temperature drought and the like.

The cultivated land area in the world is limited, and the land area of the boundary land such as saline-alkali land, drought land and the like is huge. The increase of the world population and the reduction of the arable land area seriously threaten the safety of food. In addition, with the dramatic expansion of the world population and the impact of global warming, shortages of water resources have also become one of the major challenges facing agriculture. Salinization of cultivated land and severe shortage of water resources influence sustainable development of agriculture, and research on plant salt and drought resistance mechanism has important significance on agricultural production and ecological construction.

Disclosure of Invention

The invention aims to improve the anthocyanin content and the stress resistance of plants.

The invention firstly protects the IbMYB4 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 anthocyanin content and/or stress resistance;

4) has 80 percent or more than 80 percent of homology with the amino acid sequence defined by SEQ ID NO.2, is derived from sweet potato and is a protein related to anthocyanin content and/or stress resistance.

Wherein SEQ ID NO 2 consists of 239 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 IbMYB 4.

The nucleic acid molecule encoding the protein IbMYB4 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 the protein IbMYB 4;

(a4) a DNA molecule which is derived from sweet potato and encodes the protein IbMYB4 and hybridizes 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 720 nucleotides, and the nucleotide of SEQ ID NO.1 codes an amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence of the invention encoding the protein IbMYB4 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 to the nucleotide sequence of the protein IbMYB4 isolated by the method, as long as the nucleotide sequence encodes the protein IbMYB4, are derived from and are identical to the nucleotide sequence of the method.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that has 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein IbMYB4 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-IbMYB 4. The recombinant plasmid pCB-IbMYB4 can be obtained by replacing a small fragment between recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS 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 may specifically be GV3101/pCB-IbMYB 4. The GV3101/pCB-IbMYB4 can be recombinant Agrobacterium obtained by introducing a recombinant plasmid pCB-IbMYB4 into Agrobacterium tumefaciens GV 3101.

The invention also provides application of any one of the protein IbMYB4, 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 stress resistance and/or anthocyanin content.

The invention also provides application of any one of the protein IbMYB4, 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 transgenic plants with modified stress resistance and/or anthocyanin content.

In any of the above applications, the regulating plant anthocyanin content can be increasing plant anthocyanin content.

In any of the above applications, the cultivated transgenic plant with altered anthocyanin content may be a cultivated transgenic plant with increased anthocyanin content.

In any of the above applications, the regulating plant stress resistance may be improving plant stress resistance.

In any of the above applications, the cultivation of a transgenic plant with altered stress resistance may be the cultivation of a transgenic plant with increased stress 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 IbMYB4 in a receptor plant to obtain a transgenic plant; the transgenic plant has an increased anthocyanin content and/or increased stress tolerance 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 IbMYB4 in the recipient plant can be achieved by methods 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 IbMYB4 in a recipient plant" may be specifically achieved by introducing a nucleic acid molecule encoding the protein IbMYB4 into the recipient plant.

In the above method, the nucleic acid molecule encoding the protein IbMYB4 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 IbMYB4 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-IbMYB 4. The recombinant plasmid pCB-IbMYB4 can be obtained by replacing a small fragment between recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS 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 quantity and/or activity of the protein IbMYB4 in the plant, thereby improving the anthocyanin content and/or stress 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 improvement in stress resistance can be manifested by increased survival rate, increased root length, increased fresh weight, increased SOD enzyme activity, decreased MDA content, and increased H₂O₂At least one of reduced content and increased ABA content.

Any of the above stress resistance may be salt resistance and/or drought resistance.

Experiments prove that the IbMYB4 gene is overexpressed in Arabidopsis, the anthocyanin content and the stress resistance of the Arabidopsis can be improved, and the stress resistance is improved by the following steps: increased survival rate, increased root length, increased fresh weight, increased SOD enzyme activity, decreased MDA content, and H₂O₂Reduced content and increased ABA content. Therefore, the protein IbMYB4 and the coding gene thereof can regulate and control the stress resistance and anthocyanin content of plants, and have important application value.

Drawings

FIG. 1 shows 6T₃The molecular identification result of the generation homozygous IbMYB4 transgenic Arabidopsis thaliana.

FIG. 2 shows real-time quantitative PCR detection of 6T₃The expression level of the IbMYB4 gene of the transgenic Arabidopsis IbMYB4 gene is homozygous.

FIG. 3 is T₃And (3) determining the anthocyanin content of the generation homozygous transgenic IbMYB4 gene Arabidopsis.

Fig. 4 is the result of stress resistance evaluation in step four 1 of example 2.

Fig. 5 shows the evaluation result of stress resistance in step four 2 of example 2.

FIG. 6 shows the results of measurement of physiological and biochemical parameters in step four 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.

The sweet potato high anthocyanin content 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. Hereinafter, the sweet potato high anthocyanin content strain JS6-5 is called sweet potato strain JS6-5 for short.

The vector pCAMBIA3301 is a product of Cambia corporation. The vector pBI121 is a product of Clontech. 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.

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 IbMYB4 Gene

The IbMYB4 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 O-F: 5'-ATGGTGAGGGCTCCTTGCT-3' and primer O-R: 5'-TCACGATGTATTTCCAGCATTAATA-3', obtaining a 720bp 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 IbMYB4 gene, the coded protein is named as IbMYB4 protein or protein IbMYB4, and the amino acid sequence is shown in SEQ ID NO. 2.

Example 2 application of IbMYB4 protein in regulation and control of anthocyanin synthesis and stress resistance

Construction of recombinant plasmid

1. The vector pCAMBIA3301 was double-digested with restriction enzymes HindIII and EcoRI, and the vector backbone 1 of about 11256bp was recovered.

2. The vector pBI121 was double-digested with the restriction enzymes HindIII and EcoRI, and the fragment 1 comprising about 3032bp was recovered.

3. And connecting the fragment 1 with a vector framework 1 to obtain the recombinant plasmid pCBGUS.

4. The recombinant plasmid pCBGUS was double digested with restriction enzymes BglII and PmlI, recovering vector backbone 2 of about 12388 bp.

5. 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 OE-F-Bgl II: 5' -GAAGATCTATGGTGAGGGCTCCTTGCT-3' (recognition sequence for restriction enzyme Bgl II is underlined) and primer OE-R-PmlI: 5' -GCCACGTGTCACGATGTATTTCCAGCATTAATA-3' (the recognition sequence of the restriction enzyme PmlI is underlined) and PCR amplification is carried out to obtain the double-stranded DNA molecule containing the recognition sequence of the restriction enzyme.

6. And (3) connecting the double-stranded DNA molecule containing the recognition sequence of the restriction enzyme obtained in the step (5) to a pEASY-Blunt simple vector to obtain an intermediate vector.

7. The intermediate vector was double digested with restriction enzymes BglII and PmlI, recovering fragment 2 of about 700 bp.

8. And connecting the fragment 2 with a vector framework 2 to obtain a recombinant plasmid pCB-IbMYB 4.

The recombinant plasmid pCB-IbMYB4 was sequenced. According to the sequencing result, the structure of the recombinant plasmid pCB-IbMYB4 is described as follows: the small fragment between the recognition sequences of restriction enzymes BglII and PmlI of the recombinant plasmid pCBGUS is replaced by a DNA molecule shown in SEQ ID NO.1 to obtain the recombinant plasmid. The recombinant plasmid pCB-IbMYB4 expresses IbMYB4 protein shown in SEQ ID NO. 2.

Second, obtaining of IbMYB4 transgenic Arabidopsis

1. The recombinant plasmid pCB-IbMYB4 is used for transforming agrobacterium tumefaciens GV3101 to obtain recombinant agrobacterium tumefaciens which is named as GV3101/pCB-IbMYB 4.

2. GV3101/pCB-IbMYB4 was transferred to wild type Arabidopsis thaliana by the Arabidopsis thaliana inflorescence floral dip transformation method (Clough, S.J., andBent, A.F.. Floraldip: asepli and ethephon for Agrobacterium-meditedtransformation of Arabidopsis thaliana.plant J. (1998)16, 735-₁The seed of Arabidopsis thaliana with IbMYB4 gene transfer is simulated.

3. Will T₁Planting seeds of Arabidopsis thaliana with IbMYB4 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 IbMYB4 gene positive seedling is transferred. T is₁The seeds received by the positive seedlings of the generation-transformed IbMYB4 gene are T₂Seeds of Arabidopsis thaliana with IbMYB4 gene.

4. The T of different strains screened out in the step 3₂Seeds of Arabidopsis thaliana transformed with IbMYB4 gene were sown in 1/2MS solid containing 12.5mg/LPPTScreening is carried out on a culture medium, and if the ratio of the number of arabidopsis thaliana (resistant seedlings) capable of normally growing in a certain strain to the number of arabidopsis thaliana (non-resistant seedlings) incapable of normally growing is 3: 1, the strain is a strain with an IbMYB4 gene inserted into one copy, and the seeds received by the resistant seedlings in the strain are T₃Seeds of Arabidopsis thaliana with IbMYB4 gene.

5. The T screened out in the step 4₃Seeds of arabidopsis thaliana transformed with IbMYB4 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 generation is homozygous to IbMYB4 gene Arabidopsis thaliana.

6T to be screened₃The generation homozygous IbMYB4 transgenic Arabidopsis strains are sequentially named as L1-L6.

6. Molecular identification

The arabidopsis seeds to be detected are wild arabidopsis seeds, seeds of L1, seeds of L2, seeds of L3, seeds of L4, seeds of L5 or seeds of L6.

(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 genome DNA of leaves of the arabidopsis thaliana seedlings to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of a primer O-F and a primer O-R to obtain a PCR amplification product; taking water as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer O-F and a primer O-R to obtain a PCR amplification product as a negative control; the recombinant plasmid pCB-IbMYB4 is used as a template, and a primer pair consisting of a primer O-F and a primer O-R is adopted 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 IbMYB4 transgenic arabidopsis as a template contains about 720bp DNA fragment (the same as the positive control fragment), so that the T is₃The generation homozygous IbMYB4 transgenic Arabidopsis is a positive plant; otherwise, the plant is not a positive plant.

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 showed that the PCR amplification products of L1, L2, L3, L4, L5, L6 and the positive control all contained DNA fragments of about 720bp, and the PCR amplification products of the negative control and the wild type Arabidopsis did not contain DNA fragments of about 720 bp. It can be seen that L1, L2, L3, L4, L5 and L6 are all positive plants.

7. Real-time quantitative PCR detection of T₃Expression level of generation homozygous transgenic IbMYB4 gene Arabidopsis IbMYB4 gene

(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 IbMYB4 gene in the cDNA was detected by real-time quantitative PCR (with Arabidopsis actin gene as reference gene).

The primers for detecting the IbMYB4 gene are MYB 4-F: 5'-GCGGGAAGAGTTGTAGGCTT-3' and MYB 4-R: 5'-CGAGTGTTTGGTGCAGTTGG-3' are provided.

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

The results are shown in FIG. 2. The result shows that the IbMYB4 gene is an arabidopsis thaliana foreign gene and is hardly expressed in wild arabidopsis thaliana, but the IbMYB4 gene is expressed in 6T genes₃The strains of the generation-homozygous IbMYB4 transgenic Arabidopsis have different expression degrees.

Three strains (namely L3, L5 and L6) with the highest expression level of the IbMYB4 gene are selected for subsequent experiments.

Third, determination of anthocyanin content in IbMYB4 transgenic arabidopsis

The Arabidopsis seeds to be detected are wild Arabidopsis seeds, L3 seeds, L5 seeds or L6 seeds.

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

(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 in light and dark at 22 ℃ or culturing for 5 days under pure illumination at 22 ℃ to obtain the arabidopsis thaliana seedlings to be detected.

(2) After the step (1) is completed, the Anthocyanin content of each Arabidopsis seedling to be tested is detected by referring to the method of Li and the like (Li S, Wang W, Gao J, et al. MYB75 phosphorylation by MPK4 is required for light-induced Anthocynin amplification in Arabidopsis Cell,2016,28(11): 2866-2883).

The results are shown in FIG. 3. The results show that T is compared with wild type Arabidopsis thaliana under the condition of light-dark alternate culture₃The anthocyanin content of the generation homozygous IbMYB4 transgenic arabidopsis (namely L3, L5 and L6) is obviously increased (1.2-1.5 times of that of wild arabidopsis); t is compared with wild Arabidopsis under the condition of simple light culture₃The anthocyanin content of the generation homozygous IbMYB4 transgenic arabidopsis (namely L3, L5 and L6) is remarkably increased (1.6-2.0 times of that of wild arabidopsis).

The result shows that the over-expression IbMYB4 gene can obviously promote anthocyanin synthesis of Arabidopsis.

Stress resistance identification of IbMYB4 transgenic Arabidopsis thaliana

1. Identification of stress resistance of IbMYB4 transgenic Arabidopsis thaliana

(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 completion of step (2), the Arabidopsis seedlings to be tested having substantially uniform growth vigor were transferred to a solid medium (1/2MS solid medium, 1/2MS solid medium containing 100mM NaCl, or 1/2MS solid medium containing 250mM mannitol), cultured alternately in the dark and light at 22 ℃ for 2 weeks (upright culture), and the growth state of Arabidopsis was observed.

The growth state of Arabidopsis thaliana is shown in the left panel of FIG. 4.

(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. 4.

The results showed that wild type Arabidopsis thaliana and T were cultured on 1/2MS solid medium₃The generation homozygous IbMYB4 transgenic Arabidopsis (namely L3, L5 and L6) has no significant difference in root length and fresh weight; t is compared with wild type Arabidopsis thaliana on 1/2MS solid medium containing 100mM NaCl or 1/2MS solid medium containing 250mM mannitol₃The root length and the fresh weight of the transgenic IbMYB4 Arabidopsis (namely L3, L5 and L6) are obviously increased.

2. Identification of stress resistance of IbMYB4 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;

salt stress (1 pot): culturing for 3 weeks; irrigating 50mL of 1/2 Hoagland nutrient solution containing 300mM NaCl every 2 days;

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

(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 5.

The results show that, under normal culture conditions (i.e., control), wild-type Arabidopsis thaliana and T₃The generation homozygous IbMYB4 transgenic Arabidopsis (namely L3, L5 and L6) all grow vigorously; under the condition of salt stress, the leaves of wild arabidopsis thaliana become yellow and wilted in large area, and T₃The generation homozygous IbMYB4 transgenic Arabidopsis (namely L3, L5 and L6) has only partial yellow leaves; under drought stress conditions, wild type Arabidopsis thaliana dies substantially by wilting, T₃The generation homozygous IbMYB4 transgenic Arabidopsis (namely L3, L5 and L6) has good growth state.

3. Measurement of physiological and biochemical indexes

Taking the arabidopsis seedlings to be detected which are subjected to contrast treatment for 2 weeks in the step 2 (3), the arabidopsis seedlings to be detected which are subjected to salt stress treatment for 2 weeks or the arabidopsis seedlings to be detected which are subjected to drought stress treatment for 2 weeks, detecting SOD enzyme activity by adopting a superoxide dismutase (SOD) kit (Suzhou ke Ming biology, catalog number SOD-1-Y), detecting MDA content by adopting a Malondialdehyde (MDA) kit (Suzhou ke Ming biology, catalog number MDA-2-Y), and detecting hydrogen peroxide (H)₂O₂) Kit (Suzhou ke ming bio, catalog number H)₂O₂-2-Y) detection of H₂O₂The content of the amino acid derivative is shown in the literature (ZHai H, Wang F, Si Z, et al. A myo-inositol-1-phosphate synthase, IbMIPS1, handing salt and hydrogen tolerance and stem homology resistance in transgenic sweet potato [ J]Plant Biotechnology Journal, 2016, 14(2):592.) was examined for ABA 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. 6. The results show that, under normal culture conditions (i.e., control), wild-type Arabidopsis thaliana and T₃SOD enzyme activity of transgenic IbMYB4 arabidopsis thaliana (namely L3, L5 and L6) of generation-homozygous transgenic IbMYB4 gene, MDA contentAmount H₂O₂The content and the ABA content have no obvious difference; t compared to wild type Arabidopsis under salt stress or drought stress conditions₃The SOD enzyme activities of the generation homozygous IbMYB4 transgenic arabidopsis thaliana (namely L3, L5 and L6) are all obviously increased, the MDA content is all obviously reduced, and H is₂O₂The content is obviously reduced and the ABA content is obviously increased.

The results show that the stress resistance of arabidopsis can be obviously improved by over-expressing the IbMYB4 gene; the stress resistance is salt resistance and drought resistance. The improvement of the stress resistance is shown as follows: increased survival rate, increased root length, increased fresh weight, increased SOD enzyme activity, decreased MDA content, and H₂O₂Reduced content and increased ABA content.

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

<120> sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 720

<212> DNA

<213> Ipomoea batatas

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gaagatcaaa ttttaacatc ttttatacag agatatggtc atgaaaactg gagagcctta 120

ccaagacaag ccggtttatt aaggtgcggg aagagttgta ggcttcgctg gataaattac 180

ttacgaccag atatcaaacg aggaaatttc accaaagacg aggaagaaac catcatccaa 240

ctgcaccaaa cactcggaaa cagatggtct gccattgcat caaggttgcc ggggagaaca 300

gataacgaga taaagaattt ctataacacc cacttgaaga agaggctcca gcatcatggg 360

agtccatatt attctcccaa caatgttgtc ggaaacataa cgccaatcca gatcggcgat 420

tcttcgattc acttgaggtt tccggcaccg atgactgtga actgcaatcc ggttcaccca 480

cagaacagca cgtattcaat tagctccccg atgacgacga aaatggagga agaagagagc 540

atgcaggaga gttatcaaaa ccttggaaca acatcaaacg atgattctgg gattgtctac 600

ttgccctcta gcagctctgt gttacctatg gagcttggag gttgtgagac tagttctagt 660

attagcaacg atgcagtttt ctggtataat ttgcttatta atgctggaaa tacatcgtga 720

<210> 2

<211> 239

<212> PRT

<213> Ipomoea batatas

<400> 2

Met Val Arg Ala Pro Cys Cys Glu Lys Met Gly Met Lys Lys Gly Pro

1 5 10 15

Trp Thr Pro Glu Glu Asp Gln Ile Leu Thr Ser Phe Ile Gln Arg Tyr

20 25 30

Gly His Glu Asn Trp Arg Ala Leu Pro Arg Gln Ala Gly Leu Leu Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp

50 55 60

Ile Lys Arg Gly Asn Phe Thr Lys Asp Glu Glu Glu Thr Ile Ile Gln

65 70 75 80

Leu His Gln Thr Leu Gly Asn Arg Trp Ser Ala Ile Ala Ser Arg Leu

85 90 95

Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Phe Tyr Asn Thr His Leu

100 105 110

Lys Lys Arg Leu Gln His His Gly Ser Pro Tyr Tyr Ser Pro Asn Asn

115 120 125

Val Val Gly Asn Ile Thr Pro Ile Gln Ile Gly Asp Ser Ser Ile His

130 135 140

Leu Arg Phe Pro Ala Pro Met Thr Val Asn Cys Asn Pro Val His Pro

145 150 155 160

Gln Asn Ser Thr Tyr Ser Ile Ser Ser Pro Met Thr Thr Lys Met Glu

165 170 175

Glu Glu Glu Ser Met Gln Glu Ser Tyr Gln Asn Leu Gly Thr Thr Ser

180 185 190

Asn Asp Asp Ser Gly Ile Val Tyr Leu Pro Ser Ser Ser Ser Val Leu

195 200 205

Pro Met Glu Leu Gly Gly Cys Glu Thr Ser Ser Ser Ile Ser Asn Asp

210 215 220

Ala Val Phe Trp Tyr Asn Leu Leu Ile Asn Ala Gly Asn Thr Ser

225 230 235

Claims

1. Protein IbMYB4, being 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 IbMYB4 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 percent or more homology with the nucleotide sequence limited by (a1) or (a2), is derived from sweet potato and encodes the protein IbMYB4 of claim 1;

(a4) a DNA molecule which is derived from sweetpotato and encodes the protein IbMYB4 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 IbMYB4 according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule according to claim 2 or 3, for modulating plant stress resistance and/or anthocyanin content;

b2) use of the protein IbMYB4 according to claim 1, or the nucleic acid molecule according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule according to claim 2 or 3, for the production of transgenic plants with altered stress resistance and/or anthocyanin content.

6. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the protein IbMYB4 in the receptor plant to obtain a transgenic plant; the transgenic plant has an increased anthocyanin content and/or increased stress tolerance 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 IbMYB4 in a recipient plant is realized by introducing a nucleic acid molecule encoding the protein IbMYB4 into the recipient plant.

8. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of the protein IbMYB4 in the plant, so as to improve the anthocyanin content and/or stress resistance of the plant.

9. The protein IbMYB4 of claim 1, or the use of claim 5, or the method of any one of claims 6-8, wherein: the stress resistance is salt resistance and/or drought resistance.

10. The protein IbMYB4 of claim 1, or the use of claim 5, or the method of any one of claims 6-8, 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.