Cabernet sauvignon drought-resistant gene vvdreb2a and application thereof

Abstract

The application provides a Cabernet Sauvignon grape drought-resistant gene VvDREB2A and application thereof, and the nucleotide sequence of the gene is SEQ ID NO:2. The application constructs pRI101-VvDREB2A overexpression and RNAi-VvDREB2A expression inhibition vectors, and verifies the functions by means of Agrobacterium-mediated transformation of grape callus and transient transformation of fruits. Overexpression of VvDREB2A obviously improves the drought resistance of grape callus, and increases the anthocyanin accumulation in grape callus and fruits, and inhibition of the expression affects the drought resistance of grape and the anthocyanin accumulation. The disclosed application is that the gene VvDREB2A is applied to transgenic breeding or callus factory, the drought resistance of plants is improved, and the synthesis and accumulation of grape anthocyanin are improved by regulating flavonoid pathways.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional gene screening application, and particularly relates to a Cabernet Sauvignon drought-resistant gene VvDREB2A and application thereof. BACKGROUND

[0002] Drought is considered as one of the key abiotic stress factors that have important influences on plant growth and development, leading to yield reduction of most crops and causing changes in plant morphology and physiology; in extreme cases, severe drought stress can cause plant growth stagnation and leaf wilting, and even cause plant death. In order to cope with drought stress, plants have evolved a series of complex physiological and biochemical mechanisms. Anthocyanin, as an important plant secondary metabolite, plays a key role under various environmental stresses. Its synthesis not only enhances ROS clearance and antioxidant capacity, but also effectively protects plant photosynthetic apparatus and cell components from oxidative damage, thereby significantly improving the drought resistance of plants. However, under severe drought conditions, the anthocyanin in the plant body also shows a decreasing trend.

[0003] Grape is an important economic fruit tree developed in northwest China, which can not only be eaten fresh but also be used for wine making and drying. The grape industry plays an important supporting role in the development of agricultural economy in northwest China. However, the long-term shortage of irrigation water resources is an important factor limiting the development of the grape industry. Therefore, it is an urgent need to breed grape varieties with strong drought resistance and excellent comprehensive quality in this region. Transgenic breeding has become an important direction for future agricultural breeding, which is of great significance for the rapid and directional breeding of target varieties. SUMMARY

[0004] The purpose of the present application is to provide a Cabernet Sauvignon drought-resistant gene VvDREB2A, and to prove the function of the gene in enhancing drought resistance and improving anthocyanin accumulation.

[0005] In order to achieve the above-mentioned task, the technical solution adopted by the present application is as follows:

[0006] A Cabernet Sauvignon drought-resistant gene VvDREB2A, the coding protein sequence of the gene VvDREB2A is as follows:

[0007] MSSGVIERKRKSRSRRSGPNSVAETLARWKQYNDILDSVRKAPAKGSKK

[0008] GCMKGKGGPENSICGYRGVRQRTWGKWVAEIREPNRGSRLWLGTFPTAIEA

[0009] ALAYDEAARAMYGSSARLNLPNYTTSLKDSSSAPTTSVSDSTTTTSNYSEVCA

[0010] YEDSKKPVLPSIKHESGEGESGISGGMLSAVVKAEPATPVSLVTQGGGNDPVN

[0011] VGNEPVDAMKLQHEENGHSLDAMYFKNEDGGQDFLEGFPMDEMFDVDEFL

[0012] RAIDSDPLASYGTRQELGHDSGQVGSFETDNMQWEKPTDLSYQLQNPDAKL

[0013] LGSLNHMEQVPSDFDYCYGFLQPGKQLDPCIGLNDQGLLDLELSDMGF (SEQ ID NO: 1);

[0014] The gene, one specific nucleotide sequence is as follows:

[0015] ATGTCGTCCGGAGTCATTGAAAGGAAGAGAAAGTCTCGAAGCCGGCG

[0016] AAGTGGACCTAATTCCGTGGCCGAGACTCTTGCAAGATGGAAACAATACA

[0017] ACGATATACTTGATTCTGTCCGCAAAGCTCCAGCAAAAGGTTCAAAGAAG

[0018] GGGTGTATGAAAGGTAAAGGGGGGCCCGAGAATTCAATATGTGGTTACAG

[0019] GGGTGTGAGGCAGAGGACATGGGGTAAATGGGTTGCTGAGATTCGGGAGC

[0020] CAAACAGAGGGAGTAGGCTATGGTTAGGTACCTTCCCAACTGCCATTGAA

[0021] GCTGCTCTTGCATATGACGAAGCTGCAAGGGCCATGTATGGTTCTTCTGCC

[0022] CGTCTTAATCTTCCAAATTACACCACGTCTTTGAAGGATTCTTCTTCGGCTC

[0023] CAACTACATCGGTTTCTGATTCCACCACGACAACATCCAACTACTCTGAAG

[0024] TGTGTGCATATGAGGATTCAAAGAAGCCTGTTTTACCGAGTATCAAACATG

[0025] AAAGTGGCGAAGGTGAATCAGGAATAAGTGGTGGTATGCTTTCTGCAGTG

[0026] GTTAAAGCTGAACCTGCCACACCAGTTAGTTTGGTAACACAGGGAGGAGG

[0027] TAATGATCCTGTCAATGTAGGTAATGAACCTGTTGATGCAATGAAGCTCCA

[0028] GCATGAAGAAAACGGTCATTCTTTGGATGCCATGTACTTCAAGAATGAAGA

[0029] CGGAGGACAGGATTTCTTGGAAGGATTTCCTATGGATGAAATGTTTGATGT

[0030] GGATGAATTTTTGAGGGCCATAGACTCTGACCCCCTTGCTAGCTATGGTAC

[0031] AAGGCAGGAATTGGGTCATGATTCTGGGCAAGTAGGGAGCTTTGAAACTG

[0032] ATAATATGCAGTGGGAAAAGCCAACGGATTTATCTTACCAACTGCAAAATC

[0033] CAGATGCTAAGCTTCTTGGGAGTTTGAATCATATGGAGCAAGTGCCTTCTG

[0034] ACTTTGATTATTGCTATGGCTTCTTGCAGCCAGGCAAGCAGTTAGATCCCTG

[0035] TATAGGGTTGAATGATCAGGGGTTACTTGATTTGGAATTATCAGATATGGGG

[0036] TTCTAA (SEQ ID NO: 2);

[0037] The application also provides a use of the VvDREB2A gene, which is used for enhancing drought resistance of grape.

[0038] The application further provides another use of the VvDREB2A gene, which is used for increasing anthocyanin content in grape.

[0039] The application further provides a method for screening drought-resistant grape individuals, which is achieved by detecting the expression amount of the gene.

[0040] The application finds that the growth rate of the VvDREB2A transgenic callus is obviously faster than that of the wild control, and the relative conductivity is lower than that of the control; meanwhile, the anthocyanin content in the VvDREB2A overexpression transformed callus and the transiently transformed grape fruit is significantly higher than that of the control, and the RT-qPCR analysis shows that the expression levels of the genes VvMYBA2, VvUFGT, VvCHI, VvCHS, Vv4CL and VvF3'5'H related to anthocyanin synthesis are all significantly up-regulated. The above results show that the stress resistance gene VvDREB2A of Cabernet Sauvignon grape enhances the drought resistance of the callus by overexpression, and can also promote the accumulation of anthocyanin in the grape callus and fruit. The regulation of the VvDREB2A gene expression can achieve the purposes of regulating the drought resistance of plants and changing the anthocyanin level of plants. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 1 is a diagram showing the effects of drought stress on VvDREB2A transgenic callus, wherein a shows the fresh weight determination of the grape callus treated by drought stress, and b shows the relative conductivity determination of the grape callus after drought stress treatment. Each experiment is repeated for 3 times, and different letters represent significant differences between treatments (p<0.05).

[0042] Figure 2 Figure 2 is a diagram showing the differences in anthocyanin accumulation of VvDREB2A transgenic callus after drought treatment, wherein a shows the anthocyanin content of the transgenic grape callus, and b shows the determination of the expression amount of the anthocyanin related genes of the transgenic callus. The value of PRI101 is taken as the reference and set as 1 in qRT-PCR, each experiment is repeated for 3 times, and different letters represent significant differences between treatments (p<0.05).

[0043] Figure 3Effect of transient transformation of VvDREB2A on anthocyanin accumulation in grape fruit, wherein a shows the anthocyanin content in transiently transformed grape fruit, b shows the expression of anthocyanin-related genes after transient transformation of grape fruit, qRT-PCR takes the value of empty vector pRI101 / TRV as reference and sets it as 1, each experiment is repeated 3 times, and * indicates significant difference (P<0.05) compared with the control.

[0044] Figure 4 To verify the binding of VvDREB2A to the promoters of VvMYBA2, VvCHI and VvF3'5'H, wherein a shows the binding site of VvDREB2A on the promoter of the related genes; b shows that yeast one-hybrid assay proves that VvDREB2A can interact with the promoters of VvMYBA2, VvCHI and VvF3'5'H. The yeast strain is grown on SD / -Leu and SD / -Leu+ABA medium for 3 days. The experiment is repeated 3 times, and the representative pictures are shown in the figure; c shows that EMSA (gel shift experiment) verifies the interaction between VvDREB2A and the labeled DNA probe of proVvMYBA2; d shows that dual luciferase assay shows that VvDREB2A activates the expression of proVvMYBA2::Luc in Nicotiana benthamiana leaves. DETAILED DESCRIPTION

[0045] The present application finds that the drought-related gene VvDREB2A of grape not only can enhance the drought tolerance of grape, but also has an important positive regulatory effect on the accumulation of anthocyanin, which can be applied in future grape transgenic breeding and callus factory.

[0046] The present application uses homologous cloning technology, and according to the Cabernet Sauvignon grape genome sequence, uses reverse transcription PCR (Reverse Transcription-Polymerase Chain Reaction, RT-PCR) to synthesize cDNA first strand from total RNA reverse transcription of Cabernet Sauvignon grape as a template, and first amplifies VvDREB2A in Cabernet Sauvignon grape, the complete open reading frame sequence of the gene is 1068 bp (SEQ ID NO: 2) in length, and encodes 335 amino acids (SEQ ID NO: 1).

[0047] To further study the specific function of Cabernet Sauvignon grape gene VvDREB2A, the inventors constructed pRI-VvDREB2A overexpression vector and RNAi-VvDREB2A inhibition expression vector, and expressed them in grape callus. It was found that VvDREB2A could positively regulate the drought resistance of grape callus, and the growth of the transgenic callus of pRI-VvDREB2A was faster than that of the control under drought stress, while the growth of the transgenic callus of RNAi-VvDREB2A was slower than that of the control. After drought stress treatment, the relative conductivity of the callus overexpressing VvDREB2A was lower than that of the wild type control, indicating that VvDREB2A improved the drought resistance of grape callus

[0048] In the study, the inventors determined the anthocyanin content of the transgenic callus under drought stress and the expression of anthocyanin-related genes in the transgenic callus, and the results showed that VvDREB2A could promote the accumulation of anthocyanin in grape callus.

[0049] Therefore, the present application specifically comprises the following contents:

[0050] 1. Using homologous cloning technology, the total RNA of Vitis vinifera L. cv. Cabernet Sauvignon leaves was reverse transcribed to synthesize cDNA first strand as a template, and the drought-resistant gene VvDREB2A sequence of Cabernet Sauvignon was amplified, and the coding region sequence of the drought-resistant gene VvDREB2A of Cabernet Sauvignon is SEQ ID NO: 2.

[0051] 2. pRI-VvDREB2A overexpression vector and RNAi-VvDREB2A inhibition expression vector were constructed, and they were introduced into wild type 'Jiamai' grape callus by Agrobacterium mediation. VvDREB2A transgenic lines with good performance were obtained by screening.

[0052] 3. VvDREB2A positively regulates the drought resistance of grape callus, and the growth rate of the overexpression transgenic callus is faster than that of the wild type control under drought stress, and the relative conductivity is low. In addition, the accumulation of anthocyanin in the overexpression transgenic callus and grape fruit is also significantly more than that of the wild type control, and the expression levels of VvMYBA2, VvUFGT, VvCHI, VvCHS, Vv4CL and VvF3'5'H genes related to anthocyanin synthesis are significantly up-regulated. The above results all show that the stress-resistant gene VvDREB2A of Cabernet Sauvignon grape plays an important role in up-regulating the accumulation of anthocyanin.

[0053] The present application will be described in detail below in conjunction with the examples and drawings.

[0054] Example 1: Response of VvDREB2A grape transgenic callus to drought stress

[0055] DREB2A is a kind of APETALA2 / ethylene response element binding factor type transcription factor, which is induced by drought and salt stress, in order to determine its role in grape, the CDS sequence (SEQ ID NO: 2) of Cabernet Sauvignon VvDREB2A is amplified, the overexpression vector pRI101-VvDREB2A and the suppression expression vector pCAMBIA2300-RNAi-VvDREB2A are constructed using the primers shown in Table 1 below, and the transgenic callus and the wild type callus are placed in the medium containing 20% PEG6000, and dark culture for 20 days.

[0056] Compared with the wild type callus, the VvDREB2A overexpression callus grows faster under drought stress, and the relative conductivity is lower, while the growth rate of the transgenic callus with VvDREB2A suppression expression is obviously inhibited under drought stress, and its relative conductivity is significantly higher than that of the wild type control ( Figure 1 ) These results show that VvDREB2A positively regulates the drought tolerance of grape callus.

[0057] Table 1: Sequence list of primers used in the test

[0058]

[0059] Example 2: VvDREB2A is involved in the accumulation of anthocyanins in grape callus under drought stress

[0060] Anthocyanins are an important class of plant secondary metabolites, and it is found that they also have a regulatory effect on anthocyanin synthesis during the transformation of VvDREB2A. After the transgenic callus was treated with PEG6000 to simulate drought for 20 days, the anthocyanin content of different grape callus was determined. The results show that the accumulation of anthocyanins in grape is the highest under the overexpression of VvDREB2A, while the suppression of the expression of VvDREB2A in grape callus shows the opposite phenotype ( Figure 2 A). At the same time, quantitative analysis shows that the overexpression of VvDREB2A increases the biosynthesis of anthocyanins by promoting the expression of anthocyanin biosynthesis genes including VvMYBA2, VvUFGT, VvCHI, VvCHS, Vv4CL and VvF3'5'H.

[0061] Example 3 VvDREB2A directly regulates the expression of anthocyanin synthesis related genes

[0062] In order to determine the role of VvDREB2A in plant anthocyanin synthesis, grape transient transformation was carried out at the initial stage of grape fruit coloration to verify the gene function, and the overexpression vector pRI-VvDREB2A and the viral interference vector TRV-VvDREB2A were transformed into in vitro grape fruits by Agrobacterium-mediated transformation. As shown inFigure 3 As shown in Figure A, the anthocyanin content in grapes transiently overexpressing VvDREB2A was significantly higher than that in the control, while the anthocyanin content in grapes with inhibited VvDREB2A function was lower than that in the control. VvDREB2A overexpression significantly increased the expression levels of anthocyanin synthesis pathway-related genes VvMYBA2, VvUFGT, VvCHI, VvCHS, Vv4CL, and VvF3'5'H. Figure 3 (B) VvDREB2A may affect grape anthocyanin content by regulating genes related to anthocyanin synthesis.

[0063] To further clarify the mechanism, a yeast one-hybrid (Y1H) assay was used to verify the binding of VvDREB2A to the promoters of anthocyanin synthesis-related genes. Figure 4 As shown in Figure B, after diluting the bacterial solution 1000-fold, yeast cells co-expressing pGADT7-VvDREB2A and pVvCHI-pABAi, pGADT7-VvDREB2A and pVvF3'5'H-pABAi, and pGADT7-VvDREB2A and pVvMYBA2-pABAi were able to grow on SD / -Leu+ABA medium, while the empty vector control failed to grow normally. The interaction between VvDREB2A and proVvMYBA2 was then verified by EMSA and dual-luciferase assays. These results indicate that VvDREB2A can bind to the promoters of anthocyanin synthesis-related genes VvCHI, VvF3'5'H, and VvMYBA2, thereby exerting a regulatory role.

Claims

1. A method of increasing the anthocyanin content in grapes, characterized in that, The method is to increase the expression of the gene of the amino acid sequence of SEQ ID NO: 1 in grape. The method is to increase the expression of the gene of the amino acid sequence of SEQ ID NO: 1 in grape.

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