Application of AhMYB104 gene in regulating and controlling anthocyanin content and / or saline-alkaline tolerance of plant

Abstract

The invention discloses an application of an AhMYB104 gene in regulation and control of plant anthocyanin content and / or saline-alkali tolerance, and belongs to the technical field of molecular biology. The nucleotide sequence of the AhMYB104 gene is shown as SEQ ID NO: 1, and the coded amino acid sequence of the AhMYB104 gene is shown as SEQ ID NO: 2. The AhMYB104 gene is over-expressed in arabidopsis thaliana, so that the content of anthocyanin in arabidopsis thaliana can be increased, the AhMYB104 gene in peanuts is silenced, and the tolerance of anthocyanin to saline-alkali stress can be reduced. Therefore, the AhMYB104 gene has important application value in regulating and controlling the anthocyanin content and / or saline-alkali tolerance of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to application of AhMYB104 gene in regulating anthocyanin content and / or salt-alkali tolerance of plants. Background Art

[0002] Salinization of cultivated land seriously affects crop growth and endangers food security. Discovering key genes for salt-alkali resistance is the primary task to accelerate the breeding of salt-alkali tolerant varieties. At present, the screening of salt-alkali resistance genes in various crops mainly includes WRKY, bZIP, ALDH and other family genes, and there is no report on the discovery and functional verification of MYB family genes for salt-alkali resistance.

[0003] Peanut (Arachhis hypogaea L.) is an important cash crop and oil crop, and its stable high yield is the key to ensuring the safety of edible oil. However, soil salinization has an adverse effect on the growth of peanuts, seriously reducing the yield of peanuts, especially in alkaline environments. Currently, there are few reported peanut salt-alkali resistance genes, and most of them are mainly based on salt resistance.

[0004] Therefore, developing genes that can improve plant tolerance to salt and alkali will help cultivate crop varieties that can grow normally in saline-alkali environments and increase crop yields, which is of great significance for ensuring food security and achieving sustainable agricultural development. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide an application of the AhMYB104 gene in regulating the anthocyanin content and / or salt-alkali tolerance of plants.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The use of AhMYB104 gene in regulating anthocyanin content and / or salt-alkali tolerance of plants, the amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO: 2.

[0008] Based on the above scheme, the nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO:1.

[0009] A method for regulating anthocyanin content and / or salt-alkali tolerance of plants, comprising increasing the expression level of AhMYB104 gene in the plants to increase the anthocyanin content in the plants and / or increase the salt-alkali tolerance of the plants; or decreasing the expression level of AhMYB104 gene in the plants to decrease the anthocyanin content in the plants and / or decrease the salt-alkali tolerance of the plants; the amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO: 2.

[0010] Based on the above scheme, the nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO:1.

[0011] On the basis of the above scheme, an overexpression vector or a silencing vector containing the AhMYB104 gene is constructed and transformed into a plant body, so that the AhMYB104 gene in the plant body is overexpressed or silenced, thereby increasing or decreasing the expression level of the AhMYB104 gene in the plant.

[0012] On the basis of the above scheme, the method of transforming the plant body is one of the following methods: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

[0013] Based on the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0014] Based on the above scheme, the plant is peanut or Arabidopsis thaliana.

[0015] Use of the AhMYB104 gene in preparing a product for regulating anthocyanin content and / or salt-alkali tolerance of plants, wherein the amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO: 2.

[0016] A product for regulating anthocyanin content and / or salt-alkali tolerance of plants, which is a recombinant expression vector, expression cassette, recombinant bacteria, recombinant virus or transgenic cell line containing the AhMYB104 gene sequence; the nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO: 1.

[0017] Advantages of the technical solution of the present invention

[0018] The present invention isolates a gene AhMYB104 related to anthocyanin expression from peanuts. AhMYB104 is a transcription factor that responds to salt and alkali stress, which can not only positively regulate the accumulation of anthocyanins in peanuts, but also effectively improve the tolerance of peanuts to salt and alkali stress.

[0019] Peanuts with high AhMYB104 gene expression have higher anthocyanin content than those with low AhMYB104 gene expression, and their tolerance to salt and alkali stress is also significantly higher than those with low AhMYB104 gene expression. The AhMYB104 gene was constructed into a plant overexpression vector and transformed into Arabidopsis. The anthocyanin content in Arabidopsis overexpressing the AhMYB104 gene was significantly higher than that in the wild type. The AhMYB104 gene silencing expression vector was constructed and transformed into peanuts. The anthocyanin content in the AhMYB104 gene-silenced peanuts decreased with the decrease in the relative expression of the AhMYB104 gene, and its tolerance to salt and alkali stress was also significantly reduced.

[0020] In summary, AhMYB104 gene is a gene related to the accumulation of anthocyanins and the tolerance of plants to salt and alkali stress. AhMYB104 not only helps to maintain or improve the productivity of peanuts in saline-alkali environments, which is of great significance for sustainable agricultural development; it also provides the possibility of improving the salt-alkali tolerance of peanuts and other crops through genetic engineering, which helps to expand the planting range of crops, especially in saline-alkali areas. Through the application of AhMYB104 gene, the adaptability of peanuts in changing environmental conditions can be enhanced, which has potential value in dealing with problems such as climate change and soil degradation; this is of great significance for improving crop yield and quality, protecting the agricultural ecological environment, and promoting sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 RNA-seq analysis of AhMYB genes in peanut seed coat (where BY, ZY, ZH and SH represent white, black, red and pink peanuts, respectively, and each group has 3 biological replicates; a: differentially expressed MYB genes in BY and ZY peanut seed coats; b: differentially expressed MYB genes in ZH and SH peanut seed coats; ZH4 and SH4 represent the R4 developmental stage, when peanut seeds begin to deposit pigments; ZH7 and SH7 represent the R7 developmental stage, when coloring is basically completed and close to maturity; the legend colors from green to red represent gene expression levels from low to high);

[0022] Figure 2 Peanut pods at different developmental stages (HYH, CB249, HG and HY9701 represent black, white, spotted and pink peanuts, respectively; S1 is 30 days after needle placement, S4 is 70 days after needle placement, and S7 is 110 days after needle placement);

[0023] Figure 3 Anthocyanin content of peanut seed coat at different developmental stages (HYH, CB249, HG, and HY9701 represent black, white, spotted, and pink peanuts, respectively; S1 is 30 days after needle placement, S4 is 70 days after needle placement, and S7 is 110 days after needle placement);

[0024] Figure 4 The relative expression of AhMYB104 gene in peanut germplasms with differential anthocyanin accumulation;

[0025] Figure 5 Growth status of HYH and CB249 plants after salt and alkali stress treatment (where CK represents control, SS represents salt stress treatment, and AS represents alkali stress treatment);

[0026] Figure 6 Anthocyanin content in leaves of HYH and CB249 after salt and alkali stress treatments;

[0027] Figure 7 Antioxidant enzyme activity (a), malondialdehyde content (b), and AhMYB104 gene expression (c) in peanut leaves after 5 days of salt stress;

[0028] Figure 8 Antioxidant enzyme activity (a), malondialdehyde content (b), and AhMYB104 gene expression (c) in peanut leaves after 5 days of alkaline stress;

[0029] Fig. 9 Relative expression level of AhMYB104 gene (a) and anthocyanin content (b) in AhMYB104 overexpressing Arabidopsis, where Control-1 / 2 / 3 refers to wild-type Arabidopsis, and OE-1 / 2 / 3 refers to AhMYB104 overexpressing plants;

[0030] Fig.10 Relative expression level of AhMYB104 gene (a) and anthocyanin content (b) in peanut AhMYB104 gene silenced plants, where Control-1 / 2 / 3 are wild-type plants and R-1 / 2 / 3 are AhMYB104 silenced plants.

[0031] Fig.11 Evans blue staining results of peanut leaves under alkaline stress;

[0032] Fig.12 Anthocyanin content in peanut leaves under alkaline stress;

[0033] Fig.13 The accumulation of malondialdehyde (MDA) in peanut leaves under alkaline stress;

[0034] Fig.14 Antioxidant enzyme activities in peanut leaves under alkaline stress (each group had 3 biological replicates). DETAILED DESCRIPTION

[0035] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0037] In the following embodiments,

[0038] The wild-type Arabidopsis thaliana was the Columbia wild-type Arabidopsis thaliana, which was preserved by the Shandong Peanut Research Institute.

[0039] The AhMYB104 gene silenced peanut plants were derived from Huayuhei (HYH) peanut as transgenic recipient plants, and this peanut variety was preserved by the Shandong Peanut Research Institute.

[0040] The PBI121-35S plasmid was purchased from Beijing Tianenze Gene Technology Co., Ltd. in 2019. The plasmid provided by the company is a plant binary expression vector with a CaMV35S promoter and a constitutive expression mode.

[0041] The pTRV2 plasmid was provided by Shandong Notting Biotechnology Co., Ltd. and obtained in 2024. This plasmid is a plant RNAi vector with CaMV35S promoter and kanamycin resistance.

[0042] The competent cells used for constructing the recombinant plasmids were DH5a and Agrobacterium GV3101, which were purchased from Beijing Qingke Biotechnology Co., Ltd. and Shanghai Sangon Biotechnology Co., Ltd., respectively.

[0043] In the following examples, the method for determining the expression level of the AhMYB104 gene is as follows:

[0044] The total RNA of the samples was extracted using the fastpure viral RNA min kit (Novogene, Nanjing, China), and the single-stranded cDNA was synthesized by reverse transcription using the EvoM-MLV reverse transcription premix kit (Aicore, Beijing, China). The peanut Act11 gene was used as the internal reference gene, and the SYBR Green Pro Taq HS premixed qPCR kit was used to perform qRT-PCR reaction on the ABI7500 real-time fluorescence quantitative PCR instrument to determine the expression level of the gene. -△△CT Methods The relative expression levels of genes were expressed, and three technical replicates were designed for each experiment.

[0045] The anthocyanin content was determined according to the instructions of the detection kit (R24149) provided by Shanghai Yuanye Biotechnology Co., Ltd.

[0046] The methods for determining the antioxidant enzyme activity and malondialdehyde content refer to the detection kits (BC0200, BC0090, BC0170, BC0020) provided by Beijing Solebow Biotechnology Co., Ltd. The sample extract was prepared as follows: 0.1 g of fresh peanut leaves were weighed, 1 mL of the extract was added for ice bath homogenization, centrifuged at 8000 g for 10 min at 4 °C, the supernatant was taken, and placed on ice for testing. The specific determination steps refer to the enzyme activity determination kits.

[0047] The specific method of Evans blue staining is as follows: pick peanut leaves, wash them with pure water, and wipe them dry. Soak the leaves in 0.25% (W / V) Evans blue solution for 24 hours. Then take out the leaves, wash the blue dye on the surface with pure water, absorb the water from the leaf surface, and put them in boiling anhydrous ethanol: glycerol (9:1) for 30 minutes to remove chlorophyll until the base color of the leaves appears white.

[0048] Different lowercase letters and "*" in the accompanying drawings of the present invention indicate significant differences between different groups (p<0.05).

[0049] Example 1

[0050] Cloning of AhMYB104 gene

[0051] Using black peanut "HYH" seed coat cDNA as a template, in order to ensure the specificity of the primers, primers were designed on the sequence other than the AhMYB104 gene CDS, and the AhMYB104 gene was PCR amplified using VAZYME 2×phanta Max Master Mix. The PCR amplification product was separated by 1.0% (W / V) agarose gel electrophoresis, and the target band was recovered and purified using a gel recovery kit (OmegaD2400-01, Omega, USA). The recovered target gene product was connected to the cloning vector by blunt end connection using the pEASY-Blunt Cloning Vector cloning vector kit (Quanshijin Biotechnology Co., Ltd., Beijing). The connection product was transformed into DH5a competent state (Beijing Qingke Biotechnology Co., Ltd., Beijing) by heat shock method, and the positive clone bacterial solution was selected and entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2.

[0052] SEQ ID NO:1(5'→3')

[0053] ATGGCTCCAAAGAATAATAAAAAGTCAACTAAGATATCATCAGTGATGATGAATAGAGGGGCATGGACCCCAGAAGAAGATGATAAACTAGCTCGGTTCATTGAAATTCATGGTGCAAAGAGGTGGAAGACTCTTGCAGTCAAATCAGGTCTAAAAAGATGCGGAAAAAGTTGCAGGCTGAGATGGTTAAACTATCTTAGACCCAATATCAAGAGGGGAAACATAACAATTGAAGAAGAAGACTTGATTCTTAGGCTTCACAAACTTCTAGGAAACAGATGGTCTCTGATAGCTGGGAGGCTTCCAGGGCGAACAGATAATGAAATAAAGAACCACTGGAATTCTCATTTGTGCAAAAAAGTAAATCCCAATGCAGGAGAACCATCAACTTCAACAGCAAAAGAAAGTGGTGCCACACTCAATAATATGGAGGACAGTAAAATAATGTTAGAGCATAATAGAGCCACTAATAATGGGAGTGACGAAAATTTAGATATTAACTTCGATGTGAACGAATTTTTTGATTTCTCTACTGAAGGATCCTTTGGTTTTGATTGGGCAAACAAGTACTTAGAATTTGACGAGTCATGA

[0054] SEQ ID NO:2

[0055] MAPKNNKKSTKISSVMMNRGAWTPEEDDKLARFIEIHGAKRWKTLAVKSGLKRCGKSCRLRWLNYLRPNIKRGNITIEEEDLILRLHKLLGNRWSLIAGRLPGRTDNEIKNHWNSHLCKKVNPNAGEPSTSTAKESGATLNNMEDSKIMLEHNRATNNGSDENLDINFDVNEFFDFSTEGSFGFDWANKYLEFDES

[0056] The specific primer sequences of the AhMYB104 gene are as follows:

[0057] Forward primer F: 5’-AAAAGCCTTAGTCGTCACAC-3’ (SEQ ID NO:3);

[0058] Reverse primer R: 5'-TACACATTTCGTTTCTACCAG-3' (SEQ ID NO: 4);

[0059] The PCR reaction system was: cDNA 1 μL, Mix 10 μL, Primer F+R 1 μL, ddH2O 8 μL.

[0060] The PCR reaction program was: 95°C for 3 min; 95°C for 15 sec, 55°C for 15 sec, 72°C for 30 sec, 35 cycles; 72°C for 5 min.

[0061] Example 2

[0062] Construction of AhMYB104 gene overexpression vector and recombinant strain

[0063] (1) Using a cloned plasmid containing the ORF sequence of the AhMYB104 gene as a template, PCR amplification was performed using primers containing restriction sites, and agarose gel electrophoresis, gel recovery and purification were performed to obtain the AhMYB104 gene fragment carrying the restriction site.

[0064] The primer sequences containing restriction enzyme cutting sites are as follows:

[0065] Forward primer F: 5′-gagaacacgggggactctagaATGGCTCCAAAGAATAATAAAAAGTCA-3′ (SEQ ID NO: 5);

[0066] Reverse primer R: 5′-gctcaccatggatcctctagaTGACTCGTCAAATTCTAAGTACTTGTTT-3′ (SEQ ID NO: 6);

[0067] (2) The PBI121-35S plasmid DNA was digested with QuickCutTM XbaI restriction endonuclease, and the digested product was separated, recovered and purified by electrophoresis to obtain a linearized vector fragment.

[0068] (3) The AhMYB104 gene fragment carrying the restriction site was ligated to the linearized PBI121-35S vector using ClonExpressR II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd., China). The ligation product was transformed into DH5a competent medium (Beijing Qingke Biotech Co., Ltd., China), and positive clones were selected for sequencing. The PBI121-35S-AhMYB104 overexpression vector was successfully constructed if the sequencing result was correct.

[0069] (4) The PBI121-35S-AhMYB104 overexpression vector was transformed into Agrobacterium GV3101 competent cells (Beijing Qingke Biotechnology Co., Ltd., China), and then spread onto solid LB medium containing kanamycin and rifampicin and cultured at 28°C for 2 days. After the growth of a single colony, a single clone was harvested for PCR verification, and the resulting positive clone was the successfully constructed AhMYB104 gene overexpression recombinant strain GV3101-AhMYB104; the positive clone was expanded in liquid LB medium and added with 50% glycerol at a ratio of 1:1 and stored in a -80°C refrigerator for subsequent infection.

[0070] Example 3

[0071] Construction of VIGS virus-induced silencing vector for AhMYB104 gene

[0072] (1) The CDS sequence of the AhMYB104 gene obtained in Example 1 was homologously compared with the peanut genome sequence and the NCBI database, and a highly homologous 176 bp fragment was selected to design specific primers for the AhMYB104 gene. The primer sequences are as follows:

[0073] Forward primer F: 5′-gtgagtaaggttaccgaattcATATCATCAGTGATGATGAATAGAGGG-3′ (SEQ ID NO: 7);

[0074] Reverse primer R: 5′-cgtgagctcggtaccggatccGTTCTTTATTTCATTATCTGTTCGCC-3′ (SEQ ID NO: 8);

[0075] (2) The above primers were used to amplify the highly homologous AhMYB104 gene fragment by PCR, which was reversely inserted between EcoRI and BamHI of the pTRV2 vector. The one that was correctly sequenced was the constructed AhMYB104 gene VIGS virus-induced silencing vector pTRV2-AhMYB104.

[0076] (3) The silencing vector pTRV2-AhMYB104 obtained above was transformed into Agrobacterium GV3101 competent cells (Beijing Qingke Biotechnology Co., Ltd., China), and the positive clones were screened to obtain the successfully constructed GV3101-pTRV2-AhMYB104 gene silencing strain.

[0077] Example 4

[0078] Correlation between AhMYB104 gene and anthocyanin biosynthesis in peanut

[0079] The transcriptome sequencing data of pink (SH), red (ZH), black (ZY) and white (BY) peanut seed coats were downloaded from the NCBI SRA database to analyze the expression patterns of R2R3-MYB genes in different peanut germplasms with different anthocyanin accumulation. Figure 1 As shown, the analysis found that 16 R2R3-MYB genes, including AhMYB104, were differentially expressed and highly expressed in black peanuts (ZY) with high anthocyanin content.

[0080] RT-qPCR was used to further analyze the expression of AhMYB104 gene in different developmental stages of black (HYH), white (CB249), corolla (HG), and pink (HY9701) peanuts. Figure 2-Figure 4 As shown in the figure, it was found that AhMYB104 gene was basically not expressed in white peanut (CB249), but highly expressed in black peanut (HYH). The expression pattern of AhMYB104 gene was highly consistent with the accumulation trend of anthocyanins, so AhMYB104 gene was a positive regulatory factor involved in anthocyanin synthesis.

[0081] Example 5

[0082] Analysis of salt and alkali stress tolerance in peanut varieties with significant differences in anthocyanins

[0083] Two peanut varieties, HYH and CB249, with significant differences in anthocyanin content, were treated with 100mM NaCl (SS) and 80mM NaHCO3 (AS) solutions for 5 consecutive days. The growth status of HYH and CB249 plants after salt and alkali treatment was observed. Figure 5 As shown, after salt and alkali treatment, the growth state of HYH peanut was better than that of CB249 peanut.

[0084] The content of anthocyanin in the leaves of peanut varieties HYH and CB249 was measured after 5 days of salt and alkali stress treatment. Figure 6 As shown in the figure, both salt and alkali stress can significantly induce the synthesis of anthocyanins in HYH. The anthocyanin content of HYH under alkali and salt stress was 3.96 times and 1.59 times that of the control, respectively. Alkali stress is more likely to induce the synthesis of anthocyanins than salt stress, and the accumulation of anthocyanins in HYH peanut leaves is significantly higher than that in CB249 peanuts.

[0085] After 5 days of salt and alkali stress treatment, the antioxidant enzyme activities (CAT, POD, SOD), malondialdehyde (MDA) content, and AhMYB104 gene expression in the leaves of peanut varieties HYH and CB249 were further measured. Figure 7 and Figure 8 shown.

[0086] After salt stress treatment, the CAT activity in HYH peanuts did not change significantly, while the activities of POD and SOD increased significantly; the CAT activity in CB249 peanuts decreased, while the activities of POD and SOD also increased significantly, but were significantly lower than those in HYH peanuts ( Figure 7 a); After salt stress treatment, the content of MDA in HYH and CB249 peanuts increased significantly, but the content in HYH was significantly lower than that in CB249 peanuts ( Figure 7 b); After salt stress treatment, the expression level of AhMYB104 gene in HYH did not change significantly, while the expression level of AhMYB104 gene in CB249 peanut increased significantly, but was significantly lower than that in HYH peanut ( Figure 7 (c).

[0087] After alkaline stress treatment, the activities of CAT, POD and SOD in HYH and CB249 peanuts increased significantly, but the activities of CAT and POD in HYH peanuts were significantly higher than those in CB249 peanuts ( Figure 8 a); After alkaline stress treatment, the content of MDA in CB249 peanut increased significantly, while the content of HYH did not change significantly and was significantly lower than that in CB249 peanut ( Figure 8 b); After alkaline stress treatment, the expression levels of AhMYB104 gene in HYH and CB249 peanuts increased significantly, but the expression level of AhMYB104 gene in CB249 peanuts was significantly lower than that in HYH peanuts ( Figure 8 (c).

[0088] Example 6

[0089] Obtaining Arabidopsis thaliana overexpressing AhMYB104 gene

[0090] AhMYB104 was overexpressed in Arabidopsis thaliana using the Agrobacterium-mediated floral dip method. The steps are as follows:

[0091] (1) Preparation of infection fluid for overexpressing recombinant strain GV3101-AhMYB104: Take out the LB bacterial solution of GV3101-AhMYB104 positive clone from the -80°C refrigerator, apply the bacterial solution to the LB culture plate containing kanamycin and rifampicin by streaking method, and culture at 28°C overnight. Pick a single clone and place it in 1.0mL LB liquid culture medium containing kanamycin and rifampicin, and culture it at 28°C with shaking for 24h. Transfer the above 1mL bacterial solution to 20mL LB liquid culture medium containing kanamycin and rifampicin, and culture it at 28°C with shaking for 24h. Prepare a 5% sucrose solution containing 0.03% Silwet L-77 as a resuspension solution, collect the bacterial solution by centrifugation at room temperature, and use the resuspension solution to resuspend the obtained bacterial solution to OD600 = 0.8-1.0 as an infection fluid.

[0092] (2) Preparation of AhMYB104 gene overexpressing Arabidopsis thaliana by Agrobacterium-mediated floral dipping method: Wild-type Arabidopsis thaliana was used as the transgenic recipient plant, and Arabidopsis thaliana plants with white flower buds were selected for transformation. All inflorescences were immersed in the infection solution for 60 seconds, and then the Arabidopsis thaliana plants were laid down and covered with black plastic bags to keep moisture, and placed in an incubator at 22°C. After 24 hours of treatment in the dark, the Arabidopsis thaliana was stood upright, watered normally, and cultured with a light of 12,000 Lux, and cultured in the dark for 8 hours for 16 hours of light. Total RNA was extracted from fresh Arabidopsis leaves, and the cDNA template was obtained using the Thermo Scientific RevertAid first-strand cDNA synthesis kit. The 35S primer sequence of the PBI121-35S plasmid was used as the upstream primer of the PCR reaction, and the target fragment amplification primer with a restriction site was used as the downstream primer of the PCR reaction. After the PCR reaction, the target band was collected and recovered by gel recovery. The recovered product was sent to Qingke Biotechnology Co., Ltd. for sequencing to screen the positive Arabidopsis plants overexpressing the AhMYB104 gene.

[0093] The expression level of AhMYB104 gene and anthocyanin content in AhMYB104 gene overexpressed Arabidopsis were determined. Fig. 9 As shown, the expression level of AhMYB104 in the overexpression lines (OE1, OE2, and OE3) was significantly increased compared with the wild type (Control). Fig. 9 At the same time, the anthocyanin content of the leaves of the overexpression lines (OE1, OE2, and OE3) was approximately 2.76 times that of the wild type ( Fig. 9 (b) This indicates that overexpression of AhMYB104 can promote the accumulation of anthocyanins in Arabidopsis and that AhMYB104 plays a key role in the biosynthesis of anthocyanins.

[0094] Example 7

[0095] Construction of peanut AhMYB104 gene silenced plants

[0096] The steps for using virus to induce peanut AhMYB104 gene silencing are as follows:

[0097] (1) Take out the LB culture medium of the positive clone of GV3101-pTRV2-AhMYB104 from the -80℃ refrigerator, and use the streak method to spread the culture medium on the LB culture plate containing kanamycin and rifampicin, and culture it at 28℃ overnight. Pick a single clone and place it in 1.0mL LB liquid culture medium containing kanamycin and rifampicin, and culture it at 28℃ with shaking for 24h. Transfer the above 1mL of culture medium to 20mL LB liquid culture medium containing kanamycin and rifampicin, and culture it at 28℃ with shaking for 24h. The LB culture medium of the positive clone of GV3101-pTRV1 was revived and expanded according to the above method. A solution containing 10 mM MES, 10 mM magnesium chloride, and 200 μmol / L acetosyringone was prepared as a resuspension solution, and the GV3101-pTRV2-AhMYB104 and GV3101-pTRV1 bacterial liquids were collected by centrifugation at room temperature, and the obtained bacterial liquids were resuspended to OD600 = 0.8-1.0 using the resuspension solution, and the obtained GV3101-pTRV2-AhMYB104 and GV3101-pTRV1 resuspensions were mixed in a volume ratio of 1:1, and then placed in the dark at room temperature for 60 min as an infection solution.

[0098] (2) HYH black peanut was used as the transgenic recipient plant, and peanut seedlings with the third true leaf unfolded were selected for transformation. The peanut seedlings were infected by vacuum dipping, and all peanut seedlings were immersed in the infection solution and soaked under vacuum for 5 minutes. Then, the peanut seedlings were transferred to Holland nutrient solution for culture, covered with a black plastic bag, and placed in a greenhouse at 28°C. After 24 hours of treatment in the dark, they were cultured under normal light, and cultured in the dark for 8 hours after 16 hours of light.

[0099] qPCR analysis of the expression level of AhMYB104 in peanut AhMYB104 gene silenced plants, such as Fig.10 As shown in a, compared with the wild type (Control), the expression level of AhMYB104 in the silenced plants (R-1, R-2 and R-3) was significantly downregulated, and the anthocyanin content was significantly reduced by about 26.40% ( Fig.10 (b) The results are consistent with those of the overexpression experiment, indicating that AhMYB104 is involved in the positive regulation of peanut anthocyanin synthesis.

[0100] Example 8

[0101] Tolerance of peanut AhMYB104 gene silenced plants to alkaline stress

[0102] The wild-type and silenced plants were treated with 80 mM NaHCO3 solution for 5 consecutive days. The integrity and cell activity of leaf cells were evaluated by Evans blue staining. Fig.11 As shown, under alkaline stress, the cell integrity and cell activity of peanut leaves in silenced plants were lower than those in wild-type plants.

[0103] The anthocyanin content of peanut leaves after alkaline stress was measured. Fig.12 As shown, the anthocyanin level in AhMYB104-silenced plants was lower than that in wild-type plants.

[0104] The malondialdehyde accumulation and antioxidant enzyme activity of peanut leaves after alkaline stress were measured. Fig.13 and Fig.14 As shown, Fig.14 The three columns of the same color represent three biological replicates; the accumulation of malondialdehyde in the silenced plants was higher than that in the wild-type plants, while the antioxidant enzyme activity in the wild-type plants was higher than that in the silenced plants, indicating that AhMYB104 may confer tolerance to alkaline stress in peanut by regulating the accumulation of anthocyanins.

[0105] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. The use of AhMYB104 gene in regulating anthocyanin content and / or salt-alkali tolerance of plants, characterized in that: The amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO:

2.

2. The use of the AhMYB104 gene in regulating anthocyanin content and / or salt-alkali tolerance of plants according to claim 1, characterized in that: The nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO:

1.

3. A method for regulating anthocyanin content and / or salt-alkali tolerance of plants, characterized in that: By increasing the expression level of the AhMYB104 gene in the plant, the anthocyanin content in the plant and / or the salt-alkali tolerance of the plant is increased; or by reducing the expression level of the AhMYB104 gene in the plant, the anthocyanin content in the plant and / or the salt-alkali tolerance of the plant is reduced; the amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO:

2.

4. The method for regulating plant anthocyanin content and / or salt-alkali tolerance according to claim 3, characterized in that: The nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO:

1.

5. The method for regulating plant anthocyanin content and / or salt-alkali tolerance according to claim 3, characterized in that: An overexpression vector or a silencing vector containing the AhMYB104 gene is constructed and transformed into a plant body, so that the AhMYB104 gene in the plant body is overexpressed or silenced, thereby increasing or decreasing the expression level of the AhMYB104 gene in the plant.

6. The method for regulating anthocyanin content and / or salt-alkali tolerance of plants according to claim 5, characterized in that: The method of transforming the plant body is one of Agrobacterium-mediated method, gene gun method, electric shock method, PEG method and liposome method.

7. The method for regulating anthocyanin content and / or salt-alkali tolerance of plants according to claim 6, characterized in that: The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

8. The method for regulating anthocyanin content and / or salt-alkali tolerance of plants according to any one of claims 3 to 7, characterized in that: The plant is peanut or Arabidopsis thaliana.

9. Use of the AhMYB104 gene in preparing a product for regulating anthocyanin content and / or salt-alkali tolerance in plants, characterized in that: The amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO:

2.

10. A product for regulating anthocyanin content and / or salt-alkali tolerance of plants, characterized in that: It is a recombinant expression vector, expression cassette, recombinant bacteria, recombinant virus or transgenic cell line containing the AhMYB104 gene sequence; the nucleic acid sequence of the AhMYB104 gene is shown in SEQ ID NO:1.

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