Use of ahmyb104 gene in regulating anthocyanin content and / or salt and alkali tolerance of plants

Abstract

The application discloses application of an AhMYB104 gene in regulating anthocyanin content and / or salt and alkali tolerance, and belongs to the technical field of molecular biology.The nucleic acid sequence of the AhMYB104 gene is shown as SEQ ID NO:1, and the amino acid sequence coded by the AhMYB104 gene is shown as SEQ ID NO:2.Overexpression of the AhMYB104 gene in Arabidopsis thaliana can increase the anthocyanin content in the Arabidopsis thaliana.Silencing of the AhMYB104 gene in peanuts can reduce the anthocyanin content and the salt and alkali tolerance of the peanuts.Therefore, the AhMYB104 gene has important application value in regulating anthocyanin content and / or salt and alkali tolerance of plants.

Description

TECHNICAL FIELD

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

[0002] Soil salinization seriously affects crop growth and harms food security. It is the primary task to mine key salt and alkali resistant genes for accelerating breeding of salt and alkali resistant special varieties. At present, the screening of salt and alkali resistant genes in crops mainly includes WRKY, bZIP, ALDH and other family genes, and there is no report on mining and functional verification of salt and alkali resistant MYB family genes.

[0003] Peanut (Arachis hypogaea L.) is an important economic crop and oil crop, and its stable and high yield is the key to ensure the safety of edible oil. However, soil salinization has an adverse effect on the growth of peanuts and seriously reduces the yield of peanuts, especially in alkaline environment. There are few reported peanut salt and alkali resistant genes, and most of them are based on salt resistance as the main research basis.

[0004] Therefore, developing genes that can improve the salt and alkali tolerance of plants helps to cultivate crop varieties that can grow normally in salt and alkali environments and improve crop yield, which is of great significance to food security and sustainable agricultural development. SUMMARY

[0005] In view of the problems in the prior art, the application aims to provide application of AhMYB104 gene in regulation of anthocyanin content and / or salt and alkali tolerance of plants.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

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

[0008] On the basis of 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 and alkali tolerance of plants, by increasing the expression amount of AhMYB104 gene in plants to increase the content of anthocyanin in plants and / or improve the salt and alkali tolerance of plants; or by reducing the expression amount of AhMYB104 gene in plants to reduce the content of anthocyanin in plants and / or reduce the salt and alkali tolerance of plants; the amino acid sequence encoded by the AhMYB104 gene is shown in SEQ ID NO: 2.

[0010] On the basis of the above scheme, the nucleic acid sequence of the AhMYB104 gene is shown as SEQ ID NO: 1.

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

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

[0013] On the basis of the above scheme, the Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0014] On the basis of the above scheme, the plant is peanut or Arabidopsis.

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

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

[0017] Advantages of the technical scheme of the application

[0018] The application separates and obtains a gene AhMYB104 related to the anthocyanin expression amount from peanut, and the AhMYB104 is a transcription factor responding to salt and alkali stress, which can not only positively regulate the accumulation of anthocyanin in peanut, but also effectively improve the tolerance of peanut to salt and alkali stress.

[0019] The peanut with high AhMYB104 gene expression amount has higher anthocyanin content than the peanut with low AhMYB104 gene expression amount, and the tolerance to salt and alkali stress is also significantly higher than that of the peanut with low AhMYB104 gene expression amount. The AhMYB104 gene is constructed into a plant overexpression vector, and is transformed into Arabidopsis, and the anthocyanin content in the Arabidopsis overexpressing the AhMYB104 gene is significantly higher than that of the wild type. The AhMYB104 gene is constructed into a silencing expression vector, and is transformed into peanut, and the anthocyanin content in the peanut with silenced AhMYB104 gene is reduced with the decrease of the relative expression amount of the AhMYB104 gene, and the tolerance to salt and alkali stress is also significantly reduced.

[0020] In summary, AhMYB104 gene is related to anthocyanin accumulation and plant tolerance to salt and alkali stress. AhMYB104 not only helps to maintain or improve peanut productivity under salt and alkali stress, which is of great significance to the sustainable development of agriculture, but also provides the possibility to improve the salt and alkali tolerance of peanuts and other crops through genetic engineering, which helps to expand the planting range of crops, especially in salt and alkali areas. Through the application of AhMYB104 gene, the adaptability of peanuts in variable environmental conditions can be enhanced, which has potential value in coping with climate change and soil degradation. This is of great significance to improve crop yield and quality, protect the ecological environment of agriculture, and promote the sustainable development of agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 RNA-seq analysis of AhMYB genes in peanut seed coat (wherein 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 coat; b: differentially expressed MYB genes in ZH and SH peanut seed coat; ZH4 and SH4 represent R4 development stage, when peanut seeds start to pigment deposition; ZH7 and SH7 represent R7 development stage, when pigmentation is basically completed, close to maturity; the legend color from green to red indicates that the gene expression level from low to high);

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

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

[0024] Figure 4 Relative expression of AhMYB104 gene in peanut germplasm with different anthocyanin accumulation;

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

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

[0027] Figure 7 The antioxidant enzyme activity (a), the malondialdehyde content (b), and the expression amount of the AhMYB104 gene (c) of peanut leaf after salt stress for 5 days;

[0028] Figure 8 The antioxidant enzyme activity (a), the malondialdehyde content (b), and the expression amount of the AhMYB104 gene (c) of peanut leaf after alkali stress for 5 days;

[0029] Figure 9 The relative expression amount (a) and anthocyanin content (b) of the AhMYB104 gene in the AhMYB104 overexpression Arabidopsis, wherein Control-1 / 2 / 3 is wild-type Arabidopsis, and OE-1 / 2 / 3 is the AhMYB104 overexpression plant;

[0030] Figure 10 The relative expression amount (a) and anthocyanin content (b) of the AhMYB104 gene in the peanut AhMYB104 gene silencing plant, wherein Control-1 / 2 / 3 is wild-type plant, and R-1 / 2 / 3 is the AhMYB104 silencing plant.

[0031] Figure 11 The result of Evans blue staining of peanut leaf under alkali stress;

[0032] Figure 12 The anthocyanin content of peanut leaf under alkali stress;

[0033] Figure 13 The malondialdehyde (MDA) accumulation of peanut leaf under alkali stress;

[0034] Figure 14 The antioxidant enzyme activity of peanut leaf under alkali stress (wherein 3 biological replicates are respectively set in each group). DETAILED DESCRIPTION

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

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

[0037] In the following examples,

[0038] Wild-type Arabidopsis thaliana is Columbia wild-type Arabidopsis thaliana, which is preserved by Shandong Peanut Research Institute.

[0039] AhMYB104 gene-silenced peanut plants are transgenic receptor plants of HYH peanut, which is a peanut variety preserved by Shandong Peanut Research Institute.

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

[0041] pTRV2 plasmid was provided by Shandong Nuoding Biological Technology Co., Ltd. in 2024. The plasmid is a plant RNAi vector, CaMV35S promoter, and has kanamycin resistance.

[0042] The competent cells used for constructing recombinant plasmids are DH5a and Agrobacterium GV3101, which are purchased from Beijing Qikexing Biological Technology Co., Ltd. and Shenguo Biological Engineering Shanghai Co., Ltd., respectively.

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

[0044] Total RNA of the sample to be tested is extracted using fastpure viral RNA min kit (Novozyme, Nanjing, China), and single-stranded cDNA is synthesized by reverse transcription using EvoM-MLV reverse transcription premix kit (Aikewei, Beijing, China). The expression level of the gene is determined by qRT-PCR reaction using ABI7500 real-time fluorescent quantitative PCR instrument, with peanut Act11 gene as the internal reference gene and SYBR Green Pro Taq HS premix qPCR kit. The relative expression level of the gene is determined by 2 -△△CT The method represents the relative expression level of the gene, and each experiment is designed with 3 technical replicates.

[0045] The anthocyanin content determination method is carried out according to the instructions of the detection kit (R24149) provided by Shanghai Yuanye Biological Technology Co., Ltd.

[0046] The antioxidant enzyme activity and malondialdehyde content determination methods are carried out according to the detection kits (BC0200, BC0090, BC0170, BC0020) provided by Beijing Solabio Biological Technology Co., Ltd. The sample extraction solution is prepared as follows: 0.1 g of fresh peanut leaves is weighed, 1 mL of extraction solution is added for ice bath homogenization, 8000g 4℃ centrifugation for 10 min, the supernatant is taken and placed on ice for testing. The specific determination steps are referred to the enzyme activity determination kit.

[0047] The Evans blue staining method is as follows: peanut leaves are taken and washed with purified water and dried. The leaves are soaked in 0.25% (W / V) Evans blue solution for 24 h. Then the leaves are taken out, the surface blue dye is washed with purified water, and after the surface water is absorbed, the leaves are placed in boiling anhydrous ethanol: glycerol (9:1) for 30 min until the chlorophyll is removed and the leaf background color is white.

[0048] Different lowercase letters and "*" in the figures of the present application represent significant differences between groups p < 0.05.

[0049] Example 1

[0050] Cloning of AhMYB104 gene

[0051] Taking black peanut "HYH" seed coat cDNA as a template, in order to ensure the specificity of the primer, the primer is designed on the sequence outside the CDS of AhMYB104 gene, and VAZYME 2x phanta Max Master Mix is used for PCR amplification of AhMYB104 gene. The PCR amplification product is separated by 1.0% (W / V) agarose gel electrophoresis, and the target band is recovered and purified using a gel recovery kit (Omega D2400-01, Omega, USA). The recovered target gene product is connected to the cloning vector by means of blunt end ligation using a pEASY-Blunt Cloning Vector cloning vector kit (Quansky Biotechnology Co., Ltd., Beijing). The ligation product is transformed into DH5a competent cells (Beijing Qikexin Biotechnology Co., Ltd., Beijing) by heat shock method, and positive clone bacteria liquid is picked to Beijing Qikexin Biotechnology Co., Ltd. for sequencing. The nucleic acid sequence of AhMYB104 gene is shown as SEQ ID NO: 1, and the encoded amino acid sequence is shown as SEQ ID NO: 2.

[0052] SEQ ID NO: 1 (5'→3')

[0053] ATGGCTCCAAAGAATAATAAAAAGTCAACTAAGATATCATCAGTGATGATGAATAGAGGGGCATGGACCCCAGAAGAAGATGATAAACTAGCTCGGTTCATTGAAATTCATGGTGCAAAGAGGTGGAAGACTCTTGCAGTCAAATCAGGTCTAAAAAGATGCGGAAAAAGTTGCAGGCTGAGATGGTTAAACTATCTTAGACCCAATATCAAGAGGGGAAACATAACAATTGAAGAAGAAGACTTGATTCTTAGGCTTCACAAACTTCTAGGAAACAGATGGTCTCTGATAGCTGGGAGGCTTCCAGGGCGAACAGATAATGAAATAAAGAACCACTGGAATTCTCATTTGTGCAAAAAAGTAAATCCCAATGCAGGAGAACCATCAACTTCAACAGCAAAAGAAAGTGGTGCCACACTCAATAATATGGAGGACAGTAAAATAATGTTAGAGCATAATAGAGCCACTAATAATGGGAGTGACGAAAATTTAGATATTAACTTCGATGTGAACGAATTTTTTGATTTCTCTACTGAAGGATCCTTTGGTTTTGATTGGGCAAACAAGTACTTAGAATTTGACGAGTCATGA

[0054] SEQ ID NO: 2

[0055] MAPKNNKKSTKISSVMMNRGAWTPEEDDKLARFIEIHGAKRWKTLAVKSGLKRCGKSCRLRWLNYLRPNIKRGNITIEEEDLILRLHKLLGNRWSLIAGRLPGRTDNEIKNHWNSHLCKKVNPNAGEPSTSTAKESGATLNNMEDSKIMLEHNRATNNGSDENLDINFDVNEFFDFSTEGSFGFDWANKYLEFDES

[0056] The specific primer sequence of the AhMYB104 gene is 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] PCR reaction system: cDNA 1 μL, Mix 10 μL, Primer F+R 1 μL, ddH2O 8 μL.

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

[0061] Example 2

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

[0063] (1) The cloning plasmid containing the ORF sequence of AhMYB104 gene was used as a template, and PCR amplification was performed using primers containing enzyme cutting sites. The AhMYB104 gene fragment carrying enzyme cutting sites was obtained by agarose gel electrophoresis, gel recovery and purification.

[0064] The primer sequence containing enzyme cutting sites is 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 QuickCut™ XbaI restriction endonuclease, and the digested product was electrophoretically separated, recovered and purified to obtain a linearized vector fragment.

[0068] (3) The AhMYB104 gene fragment carrying enzyme cutting sites was ligated with the linearized PBI121-35S vector using ClonExpressR II One Step Cloning Kit (Nanjing NoviGene Bio- tech Co., Ltd., China). The ligation product was transformed into DH5a competent cells (Beijing GenScript Biotech Co., Ltd., China), and positive clones were selected for sequencing. The correct sequencing result indicates that the PBI121-35S-AhMYB104 overexpression vector is successfully constructed.

[0069] (4) Transform PBI121-35S-AhMYB104 overexpression vector into Agrobacterium GV3101 competent cells (Beijing Qikexing Biotechnology Co., Ltd., China), then coat on solid LB medium containing kanamycin and rifampicin, and incubate at 28°C for 2 days. After single colony growth, harvest single clones for PCR verification, and the resulting positive clones are the successfully constructed AhMYB104 gene overexpression recombinant strain GV3101-AhMYB104; the positive clones are expanded in liquid LB medium and stored in a -80°C refrigerator with 1:1 50% glycerol for subsequent infection.

[0070] Example 3

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

[0072] (1) Homologous alignment of the CDS sequence of the AhMYB104 gene obtained in Example 1 in the peanut genome sequence and the NCBI database, selection of a highly homologous 176 bp fragment to design specific primers for the AhMYB104 gene, and 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) PCR amplification of the highly homologous AhMYB104 gene fragment using the above primers, and inserting it in reverse between EcoRI and BamHI of the pTRV2 vector. The correct sequencing is the AhMYB104 gene VIGS virus-induced silencing vector pTRV2-AhMYB104 constructed and obtained.

[0076] (3) Transform the above obtained silencing vector pTRV2-AhMYB104 into Agrobacterium GV3101 competent cells (Beijing Qikexing Biotechnology Co., Ltd., China), and the positive clones are screened to be the successfully constructed GV3101-pTRV2-AhMYB104 gene silencing strain.

[0077] Example 4

[0078] Correlation between AhMYB104 gene and anthocyanin synthesis in peanuts

[0079] The R2R3-MYB gene expression patterns in different peanut germplasms with different anthocyanin accumulation were analyzed by downloading the peanut skin transcriptome sequencing data of pink (SH), red (ZH), black (ZY) and white (BY) peanuts from the NCBI SRA database. The results are shown in Figure 1 , and it was found that 16 R2R3-MYB genes including AhMYB104 were differentially expressed, and highly expressed in black peanuts (ZY) with high anthocyanin content.

[0080] The expression of AhMYB104 gene in black (HYH), white (CB249), corolla (HG), pink (HY9701) peanuts at different development stages was further analyzed by RT-qPCR, and the results are shown in Figures 2-4 , it was found that AhMYB104 gene was not expressed in white peanuts (CB249), but highly expressed in black peanuts (HYH). The expression pattern of AhMYB104 gene was highly consistent with the accumulation trend of anthocyanin, and therefore, AhMYB104 gene was a positive regulatory factor involved in anthocyanin synthesis.

[0081] Example 5

[0082] Salt and alkali stress analysis of peanut varieties with significant differences in anthocyanin

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

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

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

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

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

[0088] Example 6

[0089] Obtaining Arabidopsis thaliana with AhMYB104 gene overexpression

[0090] AhMYB104 was overexpressed in Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method, as follows:

[0091] (1) Preparation of infection solution for overexpressing recombinant strain GV3101-AhMYB104: LB broth containing GV3101-AhMYB104 positive clones was removed from a -80℃ freezer and streaked onto LB agar plates containing kanamycin and rifampin. The plates were then incubated overnight at 28℃. Single clones were picked and placed in 1.0 mL of LB liquid medium containing kanamycin and rifampin, and cultured with shaking at 28℃ for 24 h. 1 mL of the above bacterial solution was transferred to 20 mL of LB liquid medium containing kanamycin and rifampin, and cultured with shaking at 28℃ for 24 h. A 5% sucrose solution containing 0.03% Silwet L-77 was prepared as a resuspension. The bacterial solution was collected by centrifugation at room temperature. The resuspension was used to resuspend the obtained bacterial solution to an OD600 of 0.8–1.0, which was then used as the infection solution.

[0092] (2) Preparation of AhMYB104 gene overexpression Arabidopsis by Agrobacterium-mediated dip flower method: wild-type Arabidopsis was used as the transgenic receptor plant, and Arabidopsis plants with white bud were selected for transformation. The inflorescence was immersed in the bacterial solution for 60 seconds, then the Arabidopsis plant was laid down and covered with a black plastic bag for moisture retention, and placed in a 22°C incubator. After 24 hours of treatment in the dark, the Arabidopsis was straightened up, normal watering was performed, and the light intensity was 12000Lux with 16h light and 8h dark. Fresh leaves of Arabidopsis were taken to extract total RNA, and cDNA template was obtained using Thermo Scientific RevertAid first strand cDNA synthesis kit. The 35S primer sequence of PBI121-35S plasmid was used as the upstream primer of PCR reaction, and the target fragment amplification primer with enzyme digestion site was used as the downstream primer of PCR reaction. After PCR reaction, the target band was collected and gel recovery was performed, and the recovery product was sent to Genesee Biotechnology Co., Ltd. for sequencing to screen AhMYB104 gene overexpression Arabidopsis positive plants.

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

[0094] Example 7

[0095] Construction of peanut AhMYB104 gene silencing plants

[0096] Peanut AhMYB104 gene silencing was induced by virus, and the steps were as follows:

[0097] (1) The LB culture of the GV3101-pTRV2-AhMYB104 positive clone was taken out of the -80℃ freezer and spread on LB agar plates containing kanamycin and rifampin using the streak method. It was then incubated overnight at 28℃. Single clones were picked and placed in 1.0 mL of LB liquid medium containing kanamycin and rifampin, and incubated with shaking at 28℃ for 24 h. The remaining 1 mL of the culture was transferred to 20 mL of LB liquid medium containing kanamycin and rifampin, and incubated with shaking at 28℃ for 24 h. The LB culture of the GV3101-pTRV1 positive clone was revived and expanded using the same method. A resuspension containing 10 mM MES, 10 mM magnesium chloride, and 200 μmol / L acetylsylgenone was prepared. GV3101-pTRV2-AhMYB104 and GV3101-pTRV1 bacterial suspensions were collected by centrifugation at room temperature. The obtained bacterial suspensions were resuspended to OD600 = 0.8-1.0. The obtained GV3101-pTRV2-AhMYB104 and GV3101-pTRV1 resuspensions were mixed at a 1:1 volume ratio and then incubated in the dark at room temperature for 60 min as the infection solution.

[0098] (2) Using HYH black peanut as the transgenic recipient plant, peanut seedlings with the third true leaf unfolded were selected for transformation. The peanut seedlings were infected using the vacuum immersion method. The entire peanut seedling was 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 black plastic bags, and placed in a greenhouse at 28℃. After treatment in darkness for 24 hours, they were cultured under normal light for 16 hours and then in the dark for 8 hours.

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

[0100] Example 8

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

[0102] Wild-type and silent plants were treated with 80 mM NaHCO3 solution for 5 consecutive days. Evans blue staining was used to evaluate leaf cell integrity and cell viability; the results are as follows: Figure 11 As shown, under alkaline stress, the cell integrity and cell activity of peanut leaves in silent plants were lower than those in wild-type plants.

[0103] The anthocyanin content of peanut leaves after alkali stress was determined, and the results are shown in Table 2. Figure 12 As shown in Table 2, the anthocyanin level of the AhMYB104 silenced plants was lower than that of the wild plants.

[0104] The malondialdehyde accumulation and antioxidant enzyme activity of peanut leaves after alkali stress were determined, and the results are shown in Tables 3 and 4. Figure 13 Figure 14 As shown in Tables 3 and 4, Figure 14 three biological replicates are represented by three columns of the same color; the malondialdehyde accumulation of the silenced plants was higher than that of the wild type plants, and the antioxidant enzyme activity of the wild type plants was higher than that of the silenced plants, which indicates that AhMYB104 can confer alkali stress tolerance to peanuts by regulating anthocyanin accumulation.

[0105] The above merely describes preferred embodiments of the present application, but does not limit the present application in other forms, and any person skilled in the art can modify or change the above disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution of the present application, according to the technical essence of the present application, still falls within the protection scope of the technical solution of the present application.​

Claims

1. AhMYB104 Use of a gene in increasing anthocyanin content of a plant and / or increasing salt and alkali tolerance of a plant, characterized in that, By overexpressing AhMYB104 a gene to improve the anthocyanin content of a plant and / or to improve the salt and alkali tolerance of a plant; the AhMYB104 amino acid sequence encoded by the gene is shown as SEQ ID NO: 2; and the plant is peanut or Arabidopsis.

2. The method of claim 1 AhMYB104 Use of the gene in increasing anthocyanin content of plants and / or increasing salt and alkali tolerance of plants, characterized in that, The nucleic acid sequence of the gene is set forth in SEQ ID NO:

1. AhMYB104 The nucleic acid sequence of the gene is set forth in SEQ ID NO:

1.

3. A method for modulating anthocyanin content and / or improving salt and alkaline tolerance of a plant, characterized in that, By increasing the plant AhMYB104 Increasing gene expression levels, increasing anthocyanin content in plants and / or enhancing plant tolerance to salinity; or by reducing anthocyanin levels in plants. AhMYB104 The expression level of genes reduces the anthocyanin content in plants; AhMYB104 The amino acid sequence encoded by the gene is shown in SEQ ID NO:2; the plant is peanut or Arabidopsis thaliana.

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

1. AhMYB104 The nucleic acid sequence of the gene is set forth in SEQ ID NO:

1.

5. The method for regulating anthocyanin content and / or improving salt and alkali tolerance of plants according to claim 3, characterized in that, Constructing a gene overexpression vector or a gene silencing vector, and transforming the vector into a plant body, so that the expression of the gene in the plant body is increased or decreased AhMYB104 Constructing a gene overexpression vector or a gene silencing vector, and transforming the vector into a plant body, so that the expression of the gene in the plant body is increased or decreased AhMYB104 Constructing a gene overexpression vector or a gene silencing vector, and transforming the vector into a plant body, so that the expression of the gene in the plant body is increased or decreased AhMYB104 Constructing a gene overexpression vector or 6. The method for regulating anthocyanin content and / or improving salt and alkali tolerance of plants according to claim 5, characterized in that, The transformation method of 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 improving salt and alkali tolerance of plants according to claim 6, characterized in that, The Agrobacterium is Agrobacterium tumefaciens or Agrobacterium rhizogenes.

8. AhMYB104 The use of the gene in the preparation of a product for regulating anthocyanin content and / or improving salt and alkali tolerance of plants, characterized in that, The AhMYB104 The amino acid sequence encoded by the gene is shown as SEQ ID NO: 2, and the plant is peanut or Arabidopsis.

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