CN116640197A - Application of BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress condition - Google Patents
Application of BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress condition Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to application of BhMYB79 protein in regulating plant seed germination rate and/or regulating plant growth under stress conditions. The invention provides BhMYB79 protein or application of BhMYB79 gene encoding BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress conditions, wherein the amino acid sequence of the BhMYB79 protein is shown as SEQ ID NO. 2. The BhMYB79 protein synthesized by the BhMYB79 gene disclosed by the invention is related to seed germination rate and plant growth under salt stress, the expression of the plant BhMYB79 protein is improved, or the expression of the coding gene BhMYB79 in the plant is improved, so that the seed germination rate can be reduced and the plant growth can be inhibited under the condition of salt stress.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of BhMYB79 protein in regulating plant seed germination rate and/or regulating plant growth under stress conditions.
Background
The white gourd is used as one of important vegetable crops in cucurbitaceae, has large cultivation area and high yield in China, and plays an important role in increasing income of farmers, improving agricultural efficiency and adjusting industrial structure. Most of the white gourd is cultivated in open field, so that the white gourd is greatly influenced by external environmental conditions in the growing period, which means that the white gourd can face different degrees of abiotic stress in production, including salt, drought, low temperature, high temperature and other stresses, and the yield and quality of white gourd fruits are seriously influenced.
Numerous studies have shown that MYB genes are involved in a variety of biological processes, mainly including: regulating growth and development, participating in secondary metabolism, responding to abiotic stress, etc., wherein the role of 2R-MYB genes in responding to abiotic stress such as salt, drought, low temperature, high temperature, etc. has been widely studied.
However, it is not clear at present what functions the MYB gene has when white gourd is subjected to different degrees of abiotic stress. Therefore, the existing genome and molecular biotechnology of the white gourd are utilized to excavate the white gourd 2R-MYB gene related to abiotic stress, and the preliminary exploration of the function of the gene is of great significance, so that a theoretical foundation can be laid for the healthy and stable development of the white gourd industry.
Disclosure of Invention
The invention aims to provide an application of BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress, and BhMYB79 gene encoding BhMYB79 protein can reduce seed germination rate and inhibit plant growth after overexpression.
The invention provides BhMYB79 protein or application of BhMYB79 gene encoding BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress conditions; the amino acid sequence of the Bh MYB79 protein is shown as SEQ ID NO. 2; the nucleotide sequence of BhMYB79 gene for encoding BhMYB79 protein is shown as SEQ ID NO. 1.
Preferably, the use comprises reducing the germination rate of plant seeds and/or inhibiting plant growth under stress conditions by upregulating expression of the BhMYB79 protein or upregulating expression of the BhMYB79 gene.
Preferably, the stress comprises salt stress.
Preferably, the salt stress comprises sodium chloride stress, and the working concentration of sodium chloride in the sodium chloride stress is 100-200 mM.
Preferably, the plant comprises arabidopsis thaliana or wax gourd.
The invention provides a method for constructing a model for reducing the germination rate of plant seeds and/or inhibiting plant growth under stress conditions, which comprises the following steps:
constructing an over-expression vector containing the coding gene BhMYB 79;
and (3) transforming the over-expression vector into a starting plant to obtain a model.
Preferably, the starting plant comprises arabidopsis thaliana or wax gourd.
The invention has the beneficial effects that: the invention provides BhMYB79 protein or application of BhMYB79 gene encoding BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress conditions, wherein the amino acid sequence of BhMYB79 protein is shown as SEQ ID NO.2, and the nucleotide sequence of BhMYB79 gene encoding BhMYB79 protein is shown as SEQ ID NO. 1. The BhMYB79 protein is synthesized by the coding of the wax gourd BhMYB79 gene, and the transgenic and functional identification prove that the BhMYB79 protein synthesized by the BhMYB79 gene is related to seed germination rate and plant growth under salt stress, the expression of the plant BhMYB79 protein is improved, or the expression of the coding gene BhMYB79 in the plant is improved, so that the seed germination rate under the salt stress condition can be reduced, and the plant growth and development can be inhibited.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the expression pattern of BhMYB79 gene after 150mM NaCl treatment (simulated salt stress) for 0h (CK), 4h, 8h, 12h and 24 h;
FIG. 2 is a diagram of BhMYB79 gene overexpression vector pSuper1300:BhMYB79-GFP vector;
FIG. 3 shows the expression level of BhMYB79 gene in BhMYB79 gene overexpressed lines, where WT represents non-transgenic wild-type Arabidopsis Colombia, #1- #7 represents 7 BhMYB79 overexpressed lines;
FIG. 4 shows seed germination rates under normal conditions and 0mM NaCl treatment (simulated salt stress), i.e., control group, for wild type Arabidopsis and BhMYB79 gene overexpression lines;
FIG. 5 shows seed germination rates under normal conditions and 100mM NaCl treatment (mimicking salt stress) for wild-type Arabidopsis and BhMYB79 gene over-expression lines;
FIG. 6 shows seed germination rates under normal conditions and 150mM NaCl treatment (mimicking salt stress) for wild-type Arabidopsis and BhMYB79 gene over-expression lines;
FIG. 7 seed germination rates of wild type Arabidopsis and BhMYB79 gene overexpressing lines under normal conditions and 200mM NaCl treatment (mimicking salt stress);
FIG. 8 shows the growth and development of wild-type Arabidopsis and BhMYB79 gene overexpressing lines #1, #6 and #7 under normal conditions and 100mM NaCl treatment.
Detailed Description
The invention provides BhMYB79 protein or application of BhMYB79 gene encoding BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress conditions; the amino acid sequence of the BhMYB79 protein is shown as SEQ ID NO. 2; the nucleotide sequence of BhMYB79 gene is shown as SEQ ID NO. 1.
In the present invention, the use preferably comprises reducing the germination rate of plant seeds and/or plant growth under stress conditions by upregulating expression of the BhMYB79 protein or upregulating expression of the BhMYB79 gene.
In the invention, the BhMYB79 protein is over-expressed in plants, so that the seed germination rate under the condition of salt stress can be reduced, and the growth and development of plants can be inhibited. In the invention, the reduction of the germination rate of plant seeds or the inhibition of plant growth is realized by the positive regulation of the expression of the coding gene BhMYB79. The invention discovers that the expression of the white gourd Bh MYB79 gene is improved, and can reduce the seed germination rate under the condition of salt stress and inhibit the growth and development of plants.
In the present invention, the coding gene BhMYB79 is preferably derived from the leaf of white gourd. The white gourd is preferably a white gourd high-generation inbred line B227. The invention preferably uses a forward primer BhMYB79-F (shown as SEQ ID NO: 3) and a reverse primer BhMYB79-R (shown as SEQ ID NO: 4) to carry out PCR amplification by taking leaf cDNA of the high-generation selfing B227 of white gourd as a template, thus obtaining the gene BhMYB79 for encoding BhMYB79 protein.
In the present invention, the amplification system for PCR amplification using leaf cDNA of the higher generation self-bred B227 of Benincasa hispida as a template is preferably 50. Mu.L, and comprises: primeSTARMaxPReix 25 mu L, cDNA template (independent concentration 10 ng/. Mu.L) 1 mu L, bhMYB79-F primer (independent concentration 10. Mu.M) 1 mu L, bhMYB79-R primer (independent concentration 10. Mu.M) 1 mu L, ddH 2 O22 μl; the PCR reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 52℃for 15s, elongation at 72℃for 30s,35 cycles, and final elongation at 72℃for 5min.
In the present invention, the modulation preferably comprises negative modulation, which preferably comprises decreasing the germination rate of plant seeds and/or inhibiting plant growth. In the invention, the reduction of the germination rate of plant seeds and/or the inhibition of plant growth is achieved by upregulating the expression of BhMYB79 protein. The BhMYB79 protein is over-expressed in plants, so that the seed germination rate under the condition of salt stress can be reduced, and the growth and development of the plants can be inhibited.
In the present invention, the stress preferably includes a salt stress, the salt stress of the present invention preferably includes a sodium chloride stress in which the sodium chloride concentration is preferably 100 to 200mM, more preferably 120 to 180mM, and even more preferably 150mM.
In the present invention, the plant preferably includes arabidopsis thaliana or wax gourd. In the embodiment of the invention, the transgenic arabidopsis plant is preferably realized by the following method: and (3) transforming the white gourd BhMYB79 gene into an Arabidopsis plant to obtain a transgenic Arabidopsis plant for inducing BhMYB79 gene expression. The means for transforming plant cells according to the invention preferably comprise an Agrobacterium transformation method, preferably using Agrobacterium-mediated plant genetic transformation. The agrobacterium transformation method is preferably a flower dipping method, the operation steps of the flower dipping method are not particularly limited, and the agrobacterium transformation method can be achieved by adopting a conventional method.
The agrobacterium strain of the invention is preferably agrobacterium tumefaciens GV3101. The BhMYB79 gene over-expression transgenic Arabidopsis plant is preferably verified by using a PCR amplification and fluorescent quantitative PCR method.
The PCR amplification parameters of the present invention are discussed above and will not be described in detail herein.
In the present invention, the fluorescent quantitative PCR system preferably comprises, in 10. Mu.L: 2xTransStart GreenqPCRSuperMix5 mu L, bhMYB-qRT-F (independent concentration of 10. Mu.M) primer 0.5 mu L, bhMYB-qRT-R (independent concentration of 10. Mu.M) primer 0.5 mu L, cDNA template (independent concentration of 10 ng/. Mu.L) 1. Mu.L and ddH 2 O3. Mu.L; the procedure for fluorescent quantitative PCR amplification preferably comprises: pre-denaturation at 95℃for 5s, renaturation at 58℃for 10s and extension at 72℃for 10s for 40 cycles.
The invention calculates the relative expression quantity of BhMYB79 gene by preferably using 2 -ΔΔCt When the BhMYB79 gene is subjected to fluorescence quantitative PCR, the white gourd gene BhUBQ is preferably used as an internal reference gene.
After verification, obtaining BhMYB79 gene over-expression transgenic Arabidopsis plant as T 0 And (3) replacing BhMYB79 gene-transferred Arabidopsis plants.
In the present invention, T is obtained 0 Transgenic BhMYB79 gene Arabidopsis plants are preferably harvested in the invention 0 Sowing the transgenic arabidopsis plant seeds on an MS culture medium containing hygromycin, and obtaining T after screening and transplanting 1 Transgenic arabidopsis plants, T 1 Harvesting the transgenic arabidopsis plants to obtain T 1 Transgenic arabidopsis seeds; t (T) 1 Sowing the transgenic arabidopsis seeds on an MS culture medium containing hygromycin, and obtaining T after screening and transplanting 2 Transgenic arabidopsis plants, T 2 Harvesting the transgenic arabidopsis plants to obtain T 2 Transgenic arabidopsis seeds; t (T) 2 The transgenic arabidopsis seeds are sown on an MS culture medium containing hygromycin to carry out phenotype identification on plants, the arabidopsis plants with large cotyledons and long root systems are screened out, and are transplanted in a matrix to be cultured to obtain a gene BhMYB79 over-expressed arabidopsis strain. The seeds of the BhMYB79 over-expressed Arabidopsis thaliana strain are continuously sown on an MS solid culture medium containing hygromycin, and whether the strain is separated or not is determined, so that a pure strain is obtained.
Compared with a wild type arabidopsis plant, the germination rate of the gene BhMYB79 over-expressed arabidopsis plant is reduced under the condition of salt stress, and the growth and development of the plant are inhibited. The screening according to the present invention preferably uses an MS medium containing 50 to 100mg/L hygromycin, more preferably an MS medium containing 50mg/L hygromycin.
The invention provides a method for constructing a model for reducing the germination rate of plant seeds and/or inhibiting plant growth under stress conditions, which comprises the following steps: constructing an over-expression vector containing the coding gene BhMYB 79; and (3) transforming the over-expression vector into a starting plant to obtain a model. The expression level of the plant model coding gene BhMYB79 is improved.
The original vector of the over-expression vector of the present invention preferably comprises pSuper1300-GFP. The invention preferably obtains BhMYB79 by cloning the nucleotide sequence of the BhMYB79 gene and adding homologous arm HindIII and KpnI enzyme cutting sites -TGA/+HindⅢ、KpnI The BhMYB79 gene cloned by the invention is preferably free of endStop codon TGA. Obtaining BhMYB79 -TGA/+HindⅢ、KpnI After the full length coding sequence, the invention preferably uses homologous recombination to bring the BhMYB79 -TGA/+HindⅢ、KpnI The full length coding sequence was ligated into the pSuper1300-GFP vector to give an over-expression vector. The invention preferably converts the over-expression vector into a starting plant to obtain a plant model. The starting plant according to the invention preferably comprises Arabidopsis thaliana or Benincase.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Expression pattern of BhMYB79 gene under salt stress treatment
Simulating salt stress by using 150mM NaCl solution, spraying and irrigating 'two-leaf one-heart' white gourd inbred line B227 seedlings, wherein the salt stress is divided into a salt treatment 0h group, a salt treatment 4h group, a salt treatment 8h group, a salt treatment 12h group and a salt treatment 24h group; each treatment group was set up with 3 replicates.
Leaf tissues were taken for 0h (CK, control), 4h, 8h, 12h and 24h, respectively. The expression quantity of BhMYB79 genes of each treatment group under salt stress treatment is analyzed by using qRT-PCR technology, and specific parameters are as follows:
(1) And (5) RNA extraction.
Total RNA of the leaves of white gourd is extracted by using a TransZol Up Plus RNA Kit plant total RNA extraction kit (full gold, ER 501-01), the integrity and quality of the total RNA are detected by 1% gel electrophoresis, and the concentration of the sample RNA is measured by using a NanDrop 2000C spectrophotometer (Thermo Scientific).
(2) cDNA synthesis.
Using the RNA extracted in step (1) as a template, the total RNA of each sample was reverse transcribed into cDNA using FastKing gDNA Dispelling RT SuperMix (day root, KR 118).
(3) And (2) using the cDNA obtained in the step (2) as a template, and performing qRT-PCR (real-time fluorescence quantitative PCR) by using primers of BhMYB79-qRT-F shown in SEQ ID NO. 3 and BhMYB79-qRT-R shown in SEQ ID NO. 4.
UsingGreen qPCR SuperMix (gold, AQ 101) real-time fluorescent quantitative PCR (qRT-PCR) analysis, qRT-PCR was performed on a CFX real-time fluorescent quantitative PCR instrument (BIO-RAD), qRT-PCR reaction system: 2xTransStart Green qPCR SuperMix5 mu L, bhMYB-qRT-F (independent concentration of 10. Mu.M) primer 0.5 mu L, bhMYB-qRT-R (independent concentration of 10. Mu.M) primer 0.5 mu L, cDNA template (independent concentration of 10 ng/. Mu.L) 1. Mu.L and ddH 2 O3. Mu.L. The qRT-PCR reaction procedure was: 95℃for 5s,58℃for 10s,72℃for 10s,40 cycles. The white gourd BhUBQ is taken as an internal reference gene, and the relative expression quantity of the gene is 2 -ΔΔCt And (5) calculating by a method.
The primer sequences are as follows:
BhMYB79-qRT-F：5'-CTGCGGTGGTGATAAAGA-3'(SEQ ID NO:3)；
BhMYB79-qRT-R：5'-GACCTCCAAATCCCTGTT-3'(SEQ ID NO:4)。
the results of the expression level of BhMYB79 gene under salt stress treatment in the control group and the treatment group are shown in Table 1 and FIG. 1. From table 1 and fig. 1, the BhMYB79 gene was significantly induced by salt stress.
TABLE 1 relative expression levels of BhMYB79 Gene in each treatment group
| Group of | Relative expression (relative expression level) | SE (Standard error) |
| CK | 1 | 0.44575899 |
| NaCl-4h | 61.73195669** | 7.978241611 |
| NaCl-8h | 24.41093664* | 5.380377282 |
| NaCl-12h | 20.69804485** | 10.25790183 |
| NaCl-24h | 9.257475006 | 5.359974154 |
Note that: "×" indicates that the difference is very significant compared to CK, and "×" indicates that the difference is significant compared to CK.
2. Obtaining the full-length sequence of the white gourd BhMYB79 gene
The Primer 5 software is used for designing amplification primers, leaf cDNA of high-generation selfing B227 of white gourd planted in greenhouse of the national academy of agricultural sciences of Guangdong province is taken as a template (the preparation method of the cDNA is the same as the step one), specific primers BhMYB79-F and BhMYB79-R are designed, and high-fidelity enzyme is utilizedMax DNA Polymerase (Takara, R045Q) was subjected to PCR amplification to obtain a BhMYB79 gene cDNA fragment. The PCR reaction system is as follows: primeSTAR Max Premix25 mu L, cDNA template (independent concentration 10 ng/. Mu.L) 1 mu L, bhMYB-F primer (independent concentration 10. Mu.M) 1 mu L, bhMYB-R primer (independent concentration 10. Mu.M) 1. Mu.L and ddH 2 O22. Mu.L. The PCR reaction procedure was: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 52℃for 15s, elongation at 72℃for 30s,35 cycles, final elongation at 72℃for 5min, then cooling to 4℃and preserving at-20 ℃.
Separating PCR product by agarose gel electrophoresis, cutting gel, recovering product by agarose gel DNA recovery kit (Protect, B518131-0100), and connecting the recovered product toOn T vector of BluntZ ero Cloning (Beijing full gold Biotechnology Co., ltd., CB 501), escherichia coli DH5 alpha competent cells were transformed, and finally positive clone bacterial liquid was selected and sent to biological engineering (Shanghai) Co., ltd for sequencing.
The nucleotide sequence of the obtained gene BhMYB79 is shown in SEQ ID No. 1; the amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 2.
The primer sequences are as follows:
BhMYB79-F：5'-ATGGTGAGAGCTCCATGTTG-3'(SEQ ID No.5)；
BhMYB79-R：5'-TCATAATATCTCTTTCAACTCAGA-3'(SEQ ID No.6)。
SEQ ID No.1：ATGGTGAGAGCTCCATGTTGTGAGAAAATGGGGCTCAAGAAAGGCCCTTGGACTCCTGAAGAAGACCATATTCTCATCAATTATATTAACCTCTATGGCCATGGAAATTGGAGAGCTCTCCCTAAACAAGCTGGTTTATTAAGGTGTGGAAAAAGTTGTAGGCTTAGATGGACAAATTACCTGAGGCCTGACATTAAACGGGGAAACTTAACCAAAGAAGAAGAGGAAACCATTATTAACCTACATGAAATGCTTGGAAACAGATGGTCGGCAATTGCAGCAAGATTACCCGGGAGAACCGACAATGAGATTAAAAACGTGTGGCACACCCATTTGAAAAAAAAGCTAAGACAAAATTACTTCACTCCACAACTTGAAAGTTACGCCGCCGTGAACCGGCCGATCTGCGGTGGTGATAAAGAAGAATTTATAGTACAACAACCCTTCAAATTTACGACATTTCAGCCGATGCAGGCCTCTGCTTCGGCTTCTTCCCCGCCCCATTGCTCCAGTGAGAGCTCCTCCATCGTCACGGCAGAGAACGGCGAGTTGTCAACAAATAACCTGCCGGAAGCCGACGATAATTTCTGGATGGAAGTTTTGGCCAGCGACCAAATCGCCACCGTGGGAAGCCAACAGGGATTTGGAGGTCAAAGTAGTCAGGGTCAGTTTGGTAATTTTACAATGGAGCAACCAATTCATGAGACTATGGATTTTTGGTATAATATTTTTGCAATAGGAGGGGATTTATCTGAGTTGAAAGAGATATTATGA。
SEQ ID No.2：MVRAPCCEKMGLKKGPWTPEEDHILINYINLYGHGNWRALPKQAGLLRCGKSCRLRWTNYLRPDIKRGNLTKEEEETIINLHEMLGNRWSAIAARLPGRTDNEIKNVWHTHLKKKLRQNYFTPQLESYAAVNRPICGGDKEEFIVQQPFKFTTFQPMQASASASSPPHCSSESSSIVTAENGELSTNNLPEADDNFWMEVLASDQIATVGSQQGFGGQSSQGQFGNFTMEQPIHETMDFWYNIFAIGGDLSELKEIL。
3. BhMYB79 gene over-expression vector construction
(1)BhMYB79 -TGA/+HindⅢ、KpnI Obtaining
PCR amplification is carried out by taking cDNA of BhMYB79 in the white gourd B227 leaves obtained in the second step as a template and a 35S-BhMYB79-F/35S-BhMYB79-R primer, cloning is carried out to obtain a full-length coding sequence (BhMYB 79) of a gene BhMYB79 without a stop codon (TGA) and adding homology arms (HindIII and KpnI enzyme cutting sites) -TGA/+HindⅢ、KpnI ). PCR amplification reaction system: primeSTARMaxPRIMIX 25. Mu. L, bhMYB79cDNA (independent concentration 10 ng/. Mu.L) 1. Mu.L, 35S-BhMYB79-F primer (independent concentration 10. Mu.M) 1. Mu.L, 35S-BhMYB79-R primer (independent concentration 10. Mu.M) 1. Mu.L and ddH 2 O22. Mu.L. The PCR amplification procedure is the same as in step two. Separating PCR amplified product by agarose gel electrophoresis, cutting gel, recovering to obtain gene BhMYB79 -TGA/+HindⅢ、KpnI 。
(2) Construction of an overexpression vector
Using homologous recombination method to obtain BhMYB79 in step (1) -TGA/+HindⅢ、KpnI Ligation into pSuper1300-GFP vector the ligated pSuper1300: bhMYB79-GFP vector is shown in FIG. 2. The method comprises the following specific steps:
usingBasic Seamless Cloning and Assembly Kit (full gold, CU 201) BhMYB79 -TGA/+HindⅢ、KpnI Ligation to linearized pSuper1300-GFP vector was performed in the following manner: 2xBasic Assembly Mix 5. Mu.L linearized pSuper1300-GFP vector 1. Mu.L BhMYB79 -TGA/+HindⅢ、KpnI 1. Mu.L and ddH 2 O3. Mu.L. The connection reaction is as follows: the reaction was carried out at 50℃for 15min. After the reaction is finished, the recombinant product is transformed into E.coli DH5 alpha competent cells, and finally positive clone bacterial liquid is selected and sent to the biological engineering Co-Ltd for sequencing.
The primer sequences are as follows:
35S-BhMYB79-F：5'-GACTCTAGAAAGCTTATGGTGAGAGCTCCATGTTGT-3'(SEQ ID No.7)；
35S-BhMYB79-R：5'-GCTCACCATGGTACCTAATATCTCTTTCAACTCAGA-3'(SEQ ID No.8)。
4. genetic transformation of Arabidopsis thaliana
The overexpression vector pSuper1300 constructed in the step three, bhMYB79-GFP plasmid is transformed into Agrobacterium tumefaciens competent GV3101 (Beijing Hua Va. Biotechnology Co., ltd., GT 707) by a freeze thawing method, and the genetic transformation of Arabidopsis is carried out by an Agrobacterium-mediated flower dipping method, wherein the background material of the Arabidopsis is Columbia.
5. Screening and identification of BhMYB79 over-expression transgenic lines
Obtaining T by screening the transgenic Arabidopsis obtained in the step four through hygromycin resistance (50 mg/L) and double identification of DNA level 0 The generation BhMYB79 over-expresses an Arabidopsis transgenic positive strain, and the identification primers are BhMYB79-F and BhMYB79-R.
T 0 Obtaining T by single plant seed collection of the Arabidopsis thaliana positive plant 1 The T1 generation seeds are numbered #1, #2, #3, #4, #5, #6 and #7, and 7 strains are used. T (T) 1 Sowing proper amount of seeds in MS solid culture medium (1% agar powder, pH value is 5.8, 50mg/L hygromycin) containing hygromycin in each strain of the generation seeds, counting the survival rate of the plants, carrying out chi-square test according to the plant separation ratio of the survival plants to the death plants, showing that the strain with the plant separation ratio of the survival plants to the death plants meeting 3:1 is a single copy insertion strain, carrying out single plant transplanting on positive plants meeting 3:1 strain, and harvesting the seeds by single plant to obtain T 2 Seed generation.
T 2 And (3) sowing a proper amount of seeds on an MS solid culture medium (phenotype) containing hygromycin for each single plant of the generation seeds, and carrying out phenotype identification after growing for about 10 days, wherein positive seedlings are large in cotyledons and long in root systems. The screened full-antibody strain is transplanted into a matrix for culture, and young leaves are collected and extracted after 2 weeks of culture. The Arabidopsis AtACT is taken as an internal reference gene, and the relative expression quantity of the gene is 2 -ΔΔCt Calculated by the method, the primers used are BhMYB79-qRT-F and BhMYB79-qRT-R, 3 strains #1 to #7 (see FIG. 3 and Table 2) with the BhMYB79 expression level significantly higher than that of the wild type are selected, WT represents non-transgenic wild type Arabidopsis thaliana, and #1, #6 and #7 represent an over-expressed strain of the gene BhMYB79 respectivelyIs tied up. Seeds of the three strains are continuously sown on MS solid culture medium containing hygromycin, and whether the three strains are separated is determined, so that pure strains and strains are obtained. Pure lines survived all on MS solid medium containing hygromycin (50 mg/L).
AtACT-qRT-F：5'-TAACAGGGAGAAGATGACTCAGATCA-3'(SEQ IDNo.9)；
AtACT-qRT-R：5'-AAGATCAAGACGAAGGATAGCATGAG-3'(SEQ IDNo.10)。
TABLE 2BhMYB79 Gene overexpression lines and BhMYB79 Gene expression levels in wild-type Arabidopsis thaliana
| Relative expression (relative expression level) | SD (Standard deviation) | |
| WT | 1 | 0.194212185 |
| #1 | 344.9795834** | 122.0038769 |
| #2 | 160.2208807** | 40.80163698 |
| #3 | 150.9796243** | 10.72903561 |
| #4 | 128.5896947** | 13.35095138 |
| #5 | 185.1054766** | 81.65191894 |
| #6 | 6125.044301** | 1337.758339 |
| #7 | 945.482288** | 207.7156447 |
Note that: "x" represents that the difference was very significant compared to wild-type WT.
6. Determination of seed germination rate of wild-type and BhMYB79 transgenic lines under salt stress
Taking 36 Arabidopsis seeds of the Arabidopsis wild type WT, the overexpression transgenic lines #1, #6 and #7 obtained in the fifth step, spreading the seeds on MS solid culture medium containing 0mM NaCl, 100mM NaCl, 150mM NaCl and 200mM NaCl, germinating in a culture room with the temperature of 25 ℃ (16 h of illumination and 8h of darkness), counting the number of germinated seeds every 1 day, and calculating the germination rate of the seeds according to the following formula. The radicle-protruding seeds were considered as germinating seeds, and 3 parallel experiments were performed per NaCl concentration treatment group.
Seed germination = (number of germinated seeds/36) ×100%
The results are shown in FIG. 4-FIG. 7 and Table 3-1-Table 3-4, the seed germination rate and WT of transgenic lines #1, #6 and #7 in which BhMYB79 is overexpressed under normal conditions are similar, and the seed germination uniformity is high; whereas seed germination rates for transgenic lines #1, #6 and #7 overexpressing BhMYB79 gene under salt stress of 100mM, 150mM and 200mM NaCl were significantly lower than WT.
TABLE 3-1 0mM NaCl group seed germination status of wild type Arabidopsis thaliana and BhMYB79 Gene overexpression lines
TABLE 3 seed germination Rate for wild type Arabidopsis thaliana and BhMYB79 Gene overexpression lines of 100mM NaCl group
As can be seen from Table 3-2, the germination rate of Arabidopsis seeds at 100mM NaCl was reduced within 11 days, and the germination rate of WT was higher than that of 3 BhMYB79 gene-overexpressing lines #1, #6, #7, and the Arabidopsis seeds of lines #1, #6, #7 were leveled with the WT plants after 6 days, as compared with the WT plants, within 2 to 5 days.
TABLE 3 seed germination Rate for wild type Arabidopsis thaliana and BhMYB79 Gene overexpression lines of the MNACl group-3150
As can be seen from tables 3-3, the germination rate of Arabidopsis seeds at 150mM NaCl was reduced within 11 days, and the germination rate of WT was higher than that of 3 BhMYB79 gene overexpressing lines #1, #6, #7, and the Arabidopsis seeds of lines #1, #6, #7 were leveled with the WT plants after 9 days, as compared with the WT plants, within 3 to 8 days.
Table 3-4200 MNaCl group of wild type Arabidopsis and BhMYB79 Gene overexpression lines seed germination Rate
As can be seen from tables 3-4, the BhMYB79 gene overexpressing lines #1, #6, #7 showed a reduced germination rate of Arabidopsis seeds at 200mM NaCl over 11 days compared to the WT plants, and the seed germination rate of WT was higher than that of the 3 BhMYB79 gene overexpressing lines #1, #6, #7, and was leveled with the WT plants only after 10 days.
8. Seedling growth and development of wild-type and BhMYB79 transgenic lines under salt stress
Arabidopsis wild-type WT, over-expressed transgenic lines #1, #6 and #7 were taken, 36 seeds each, plated on MS solid medium containing 0mM and 100mM, and grown in a 25℃medium (16 h light, 8h dark). Under salt stress-free conditions, the transgenic lines showed no significant difference in growth from the wild-type lines. However, transgenic seedlings grew slower under 100mM NaCl stress, and eventually only about 20% of transgenic seedlings survived, whereas the survival rate of the wild type strain reached 80%, see FIG. 8 and Table 4. Therefore, the seed germination rate of the plant over-expressing the wax gourd BhMYB79 gene is reduced under the condition of salt stress, and the growth and development of the plant are inhibited.
TABLE 4 seedling survival of wild type Arabidopsis and BhMYB79 Gene overexpressing lines
In conclusion, the invention constructs the white gourd BhMYB79 gene over-expression vector for the first time, and performs functional research by heterologous transformation of Arabidopsis thaliana. By measuring the germination rate of the Arabidopsis seeds under the salt stress and observing the growth and development condition of Arabidopsis plants, the germination rate of the seeds of the plants which over-express the wax gourd BhMYB79 gene is reduced under the salt stress condition, and the growth and development of the plants are inhibited.
According to the invention, the expression mode of the wax gourd part 2R-MYB gene under the condition of salt stress is analyzed for the first time, the expression quantity of the BhMYB79 gene under the condition of salt stress is obviously improved, and the transcription factor is presumed to be related to the salt tolerance of the wax gourd. Through heterologous overexpression of BhMYB79 genes in arabidopsis thaliana (Columbia), the germination rate of seeds and the growth and development of plants under salt stress are inhibited by the overexpression of BhMYB79 genes, and the BhMYB79 genes are shown to negatively regulate the salt tolerance of the plants.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
- BhMYB79 protein or application of BhMYB79 gene encoding BhMYB79 protein in regulating plant seed germination rate and/or plant growth under stress conditions; the amino acid sequence of the BhMYB79 protein is shown as SEQ ID NO. 2; the nucleotide sequence of BhMYB79 gene for encoding BhMYB79 protein is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein the use comprises reducing the germination rate of plant seeds and/or inhibiting plant growth under stress conditions by upregulating BhMYB79 protein expression or upregulating expression of the BhMYB79 gene.
- 3. The use according to claim 1 or 2, wherein the stress comprises salt stress.
- 4. The use according to claim 3, wherein the salt stress comprises sodium chloride stress, the working concentration of sodium chloride in the sodium chloride stress being 100-200 mM.
- 5. The use according to claim 1, wherein the plant comprises arabidopsis thaliana or wax gourd.
- 6. A method of constructing a model for reducing the germination rate and/or inhibiting plant growth of plant seeds under stress conditions, comprising the steps of:constructing an overexpression vector containing the coding gene BhMYB79 of claim 1;and (3) transforming the over-expression vector into a starting plant to obtain a model.
- 7. The method of claim 6, wherein the starting plant comprises arabidopsis thaliana or wax gourd.
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