A SoMYB1 gene and its use in preparing high-yield cyanidin-3-O-rutinoside transgenic plants

Abstract

The present invention provides a SoMYB1 gene and its use in preparing a high-yield cyanidin-3-O-rutinoside transgenic plant, belonging to the field of genetic engineering. The present invention finds for the first time that, compared with wild-type tobacco, the content of anthocyanin metabolites, especially cyanidin-3-O-rutinoside, in transgenic tobacco overexpressing the SoMYB1 gene is significantly increased. The SoMYB1 gene of the present invention, and its recombinant vector and recombinant bacteria can be used to increase the cyanidin-3-O-rutinoside content in plants. The method for constructing a high-yield cyanidin-3-O-rutinoside transgenic plant provided by the present invention has broad application prospects in the directional production of high-yield cyanidin-3-O-rutinoside transgenic plants.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to a method for preparing a high-yield cyanidin-3-O-rutinoside transgenic plant by overexpressing the SoMYB1 gene. Background Art

[0002] Cyanidin-3-O-rutinoside (C3R) is an important anthocyanin metabolite naturally present in plants. Studies have found that C3R has a protective effect on rat adrenal pheochromocytoma cells (PC12 cells) oxidative damage caused by H2O2, which is related to inhibiting the expression of proteins related to the cell apoptosis pathway, increasing the activity of intracellular antioxidant enzymes, clearing excessive intracellular ROS, and reducing cell apoptosis. It is a promising candidate natural drug for preventing neurodegenerative diseases (NDD) such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD).

[0003] In recent years, genetic engineering technology has made significant progress in the synthesis and extraction of plant medicinal substances, and has become an important method for obtaining medicinal substances. Among many plants, tobacco has long been used as a research and development object for genetic engineering to synthesize and extract medicinal substances due to its biological characteristics such as easy tissue manipulation, strong regeneration ability, and large growth. Since 2008, genetic engineering technology has been used to obtain drugs such as tumor cell surface proteins, ZMapp, serum protein activator TPA, cancer immune substances, and artemisinic acid for the treatment of diseases such as non-Hodgkin's lymphoma, Ebola virus, heart disease, cancer, and malaria.

[0004] So far, there is no report on the development of transgenic plants with high yield of cyanidin-3-O-rutinoside using genetic engineering technology. Summary of the invention

[0005] The first purpose of the present invention is to provide a new SoMYB1 gene, and its recombinant vector and recombinant bacteria, and for the first time found that overexpression of the SoMYB1 gene can significantly increase the content of cyanidin-3-O-rutinoside in plants.

[0006] The second object of the present invention is to provide a method for preparing a transgenic plant with high yield of cyanidin-3-O-rutinoside.

[0007] The present invention provides a SoMYB1 gene, whose CDS sequence is shown as SEQ ID No.1.

[0008] The present invention also provides a recombinant vector, which includes a gene with a CDS sequence as shown in SEQ ID No.1.

[0009] The present invention also provides a recombinant bacterium, which comprises the above-mentioned recombinant vector.

[0010] Furthermore, the recombinant bacteria is recombinant Agrobacterium.

[0011] Furthermore, the recombinant Agrobacterium is recombinant Agrobacterium tumefaciens.

[0012] The present invention also provides a method for preparing a transgenic plant with high yield of cyanidin-3-O-rutinoside, characterized in that the aforementioned SoMYB1 gene is transferred into the plant to obtain a plant expressing the SoMYB1 protein.

[0013] Furthermore, the method of transferring into plants is one of the following: Agrobacterium method, gene gun method, electroporation method, PEG-mediated method, liposome method, and calcium phosphate-DNA co-precipitation method.

[0014] Furthermore, the plant is tobacco.

[0015] The present invention also provides the use of the aforementioned SoMYB1 gene, the aforementioned recombinant vector or the aforementioned recombinant bacteria in preparing a transgenic plant with high yield of cyanidin-3-O-rutinoside.

[0016] Furthermore, the plant is tobacco.

[0017] The present invention has achieved the following beneficial effects:

[0018] The present invention firstly finds that, compared with wild-type tobacco, the content of anthocyanin metabolites, especially cyanidin-3-O-rutinoside, in transgenic tobacco overexpressing the SoMYB1 gene is significantly increased.

[0019] The SoMYB1 gene, the recombinant vector and the recombinant bacteria of the present invention can be used to increase the content of cyanidin-3-O-rutinoside in plants.

[0020] The method for constructing a high-yield cyanidin-3-O-rutinoside transgenic plant provided by the present invention has broad application prospects in the directional production of high-yield cyanidin-3-O-rutinoside transgenic plants.

[0021] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0022] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :Gel running diagram of SoMYB1 CDS amplification (M:D2000; 1-10 represents SoMYB1 CDS positive amplification).

[0024] Figure 2 :Gel screening of SoMYB1 CDS connected to pLB vector (M: D2000; 1-6 are positive bacteria).

[0025] Figure 3 : Gel run of SoMYB1-attB1-F and SoMYB1-attB2-R primer amplification (M: D2000; 1-6 are positive amplifications).

[0026] Figure 4 :Gel run diagram of SoMYB1 expression vector construction (M: D2000; 1. Bp reaction positive bacteria; 2. LR reaction positive bacteria; 3. BP reaction positive bacteria plasmid; 4. LR reaction positive bacteria plasmid).

[0027] Figure 5 :Gel run of SoMYB1 Agrobacterium transformation screening bacteria (M: D2000; 1-10 are positive bacteria). Figure 6 : PCR identification of transgenic tobacco lines overexpressing SoMYB1 (M:D2000; 1-9 are positive amplifications).

[0028] Figure 7 : Photographs of tobacco (CK: wild-type tobacco; SoMYB1: transgenic tobacco line). Figure 8 : Anthocyanin metabolite content in leaves of SoMYB1 overexpressing transgenic tobacco lines (CK: wild-type tobacco; SoMYB1: transgenic tobacco line; Anthocyanidin_12: cyanidin-3-O-rutinoside). Specific implementation plan

[0029] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0030] Example 1. Cloning of SoMYB1 gene

[0031] 1. Experimental methods

[0032] Through the MYB transcription factor known to regulate anthocyanin synthesis, homologous comparison was performed in the lilac transcriptome database to find the target gene SoMYB1 sequence.

[0033] RNA was extracted from lilac petals using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China) according to the instructions. TM 1stStrand cDNASynthesis Kit (TaKaRa, Beijing, China) was used to synthesize cDNA according to the instructions. Using cDNA as a template, primers SoMYB1-F and SoMYB1-R were designed according to the coding region of the SoMYB1 gene. The primer sequences were 5'-CTACAGATTTTCACGTGCTTATTGG-3' (SEQ ID No. 2); 5'-TGCATGTCTGATGTGTCTATGTCT-3' (SEQ ID No. 3), respectively, for PCR amplification.

[0034]

[0035] PCR reaction program: 94℃3min; 94℃30s, 58℃30s, 72℃90s, 35 cycles; 72℃5min, 4℃ storage. PCR products were detected by 2% agarose gel electrophoresis. The results are as follows: Figure 1 As shown, the amplification was successful.

[0036] The target fragment of SoMYB1 was recovered using the SanPrep column DNA gel recovery kit (Shanghai Shenggong Biotechnology Service Co., Ltd.) according to the instructions. The target fragment was connected according to the instructions of the pLB zero background rapid cloning kit (TIANGEN, China) for positive bacteria screening, and the positive bacteria were sent to Shanghai Shenggong for sequencing. After successful sequencing, the rapid plasmid extraction kit (TIANGEN, China) was used to extract the positive bacterial plasmid for the next step of expression vector construction according to the instructions. The results are shown in Figure 2By sequencing, the CDS sequence of the SoMYB1 gene was determined to be: ATGTTAAGTACACAAGTTGAATTGAGAAAAGGTGCATGGACTGAAGAAGAAGATAAACTTCTCCGGAAGTGCATTAACAAGTATGGAGAAGGAAAATGGCATCTAGTTCCTCTCAGAGCAGGGTTGAACAGATGCAGGAAGAGCTGTAGGCTGAGGTGGTTAAACTATCTCAGGCCAAATATCAAAAGAGGAGACTTTGCTCCGGATGAAGATGA TCTCATTATAAGGCTTCATAACTTGTTAGGAAACAGATGGTCACTGATATCCGGTAGAATTCCTGGAAGAACCGGCAACGACGTGAAAAACCATTGGAACACCCATCTGCAGAAGAAGGTATTGGGTCGAGAAGAGGGGAAGACGAAAGCCCAGAAAACCACCAAAACGACAATCTTGAGACCTCGACCTCGGACCTTCAAAAGAATTATTAATGGATCTTTTTCGTCATTAGATCAGAAAATAAAT AATATTCAGCTCTAACTGATCAAAGTTCAGACATTCCATCTCCATCATCGTCAAAACAAGTAGACGATGCATGCACCCAGACGCAGTGGTGGAGTAACTTGCTCGATTCTGTCGAAATTGAT GGAGAAACTGAGCCAGGATCTAAAGGAATATCTCTAGAATTGCAACAAGATGATGGCTGGAATGACTTTTCCTTGATAAGGGCATTTGGGAATTCCTGAGTTCTGAAAATATCATATGA(SEQ ID No.1).

[0037] The CDS of this gene is 705 bp.

[0038] Example 2: Construction of an expression vector for the SoMYB1 gene

[0039] 1. Experimental methods

[0040] The Gateway Cloning kit (Invitrogen, USA) was used to design primers SoMYB1-attB1-F and SoMYB1-attB2-R according to the instruction manual. The primer sequences are:

[0041] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTTAAGTAC ACAAGTTG-3' (SEQ ID No. 4),

[0042] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATATGATATTTTCAGA-3' (SEQ ID No. 5),

[0043] SoMYB1 plasmid was amplified using 2×Pfu PCR Mix (TIANGEN, China) according to the instruction manual.

[0044]

[0045] PCR reaction program: 94℃3min; 94℃30s, 58℃30s, 72℃2min, 30 cycles; 72℃5min, 4℃ storage, PCR products were detected by 2% agarose gel electrophoresis. Figure 3 As shown, the amplification was successful.

[0046] The PCR product was recovered and the SoMYB1 expression vector was constructed according to the Gateway Cloning kit (Invitrogen, USA) kit instructions. Entry vector construction (Bp reaction): SoMYB1-CDS sequence was connected to the entry vector pDONR207 to obtain pDONR207-SoMYB1; expression vector construction (LR reaction): pDONR207-SoMYB1 was recombined with the expression vector pJAM1502 to obtain the expression vector pJAM1502-SoMYB1. The results are shown in Figure 4 As shown, the construction was successful. The expression vector was transformed into E. coli DH5α competent cells, positive bacteria were picked, and the plasmid was extracted for the next step of Agrobacterium transformation.

[0047] The pJAM1502-SoMYB1 expression vector was transferred into the Agrobacterium tumefaciens strain GV3101 by freeze-thaw method. The specific method is as follows:

[0048] Take GV3101 Agrobacterium competent cells and place them on ice to dissolve. The reaction system is as follows: 1μL DNA plasmid, 100μL Agrobacterium competent cells. After fully mixing, place in an ice bath for 30 minutes, then freeze in liquid nitrogen for 5 minutes, take out and immediately place in a 37°C water bath for 5 minutes, and finally place on ice for 5 minutes. In the clean bench, add 500μL of YEP liquid culture medium (Agrobacterium culture medium) to the system, seal it and place it on a shaker at 220rpm and 28°C for 2 hours. In the clean bench, take 100μL of the shaken bacterial solution and spread it on the YEP solid culture medium containing 50mg / L kanamycin and 50mg / L rifampicin, and culture it upside down in the dark at 28°C for two days. The grown colonies were tested by PCR using primers SoMYB1-attB1-F and SoMYB1-attB2-R. The results are as follows Figure 5 As shown, the conversion was successful.

[0049] Example 3: SoMYB1 gene transformation of tobacco

[0050] 1. Experimental methods

[0051] The pJAM1502-SoMYB1 expression vector prepared in Example 2 was transformed into the tobacco genome using the Agrobacterium-mediated transformation method. The specific method is as follows:

[0052] After culturing tobacco (Nicotiana tabacum L.) aseptic tissue culture seedlings for 2-3 weeks, the leaves were cut into a length × width of about 1 cm × 1 cm, and the cut leaves were placed in a bacterial solution containing the pJAM1502-SoMYB1 expression vector for infection for 10 minutes, and the concentration of Agrobacterium was about 0.6 (OD600). After the bacterial solution on the leaves was dried with sterile absorbent paper, they were placed on a basic culture medium and cultured in the dark at 28°C for 2 days. The leaves were transferred to a basic culture medium containing 300 mg / L cephalosporin, cultured at 25°C and 2000 lx for 3-5 days, and then transferred to a basic culture medium containing 300 mg / L cephalosporin and 50 mg / L kanamycin (Diamond, Shanghai, China) for resistance screening. When the differentiated seedlings of the leaves grew to about 1.5 cm, the seedlings were transferred to MS culture medium containing 300 mg / L cephalosporin and 50 mg / L kanamycin and cultured for about 20 days (culture temperature was 25°C), and SoMYB1 gene-transformed tobacco was obtained.

[0053] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0054] Experimental Example 1 Identification and detection of transgenic tobacco overexpressing SoMYB1 gene

[0055] 1. Experimental samples

[0056] The transgenic tobacco overexpressing the SoMYB1 gene constructed in Example 3 was used as a control with wild-type tobacco (Nicotiana tabacum L.).

[0057] 2. Experimental Methods

[0058] The specific primers SoMYB1-attb1-F and SoMYB1-attb2-R were used to amplify the DNA of the transformed tobacco and identify the transgenic tobacco. The specific experimental method is as follows:

[0059]

[0060]

[0061] PCR reaction program: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 90 s, 35 cycles; 72°C for 5 min, stored at 4°C. The PCR products were detected by 2% agarose gel electrophoresis.

[0062] The anthocyanin metabolite contents in leaves of SoMYB1-overexpressing transgenic tobacco and wild-type tobacco were compared by anthocyanin targeted metabolomics assay (Maiwei Metabolism, China).

[0063] 3. Experimental results

[0064] Through PCR detection, a total of 9 transgenic tobacco lines overexpressing SoMYB1 were screened, such as Figure 6 .

[0065] Phenotypic observation of transgenic tobacco strains revealed that compared with wild-type tobacco, transgenic tobacco had significantly accumulated anthocyanins in different tissues. Wild-type tobacco had white roots, green stems, leaves, fruits, and seeds, and light pink flowers, while transgenic tobacco had purple roots, stems, leaves, flowers, fruits, and seeds. Figure 7 ).

[0066] Anthocyanin targeted metabolomics detection was performed on the leaves of transgenic tobacco lines overexpressing the SoMYB1 gene. It was found that the total amount of 49 anthocyanin metabolites detected increased from 28.6μg / g DW in wild-type tobacco to 1020.7μg / g DW, and the total amount of anthocyanin metabolites increased by about 36 times. Among these 49 anthocyanin metabolites, the content of cyanidin-3-O-rutinoside in transgenic tobacco lines was 912.7μg / g, which was about 4590 times that of wild-type tobacco, accounting for 89.4% of the total anthocyanin metabolites in transgenic tobacco lines. Figure 8 .

[0067] The above experimental results show that compared with wild-type tobacco, the content of anthocyanin metabolites, especially cyanidin-3-O-rutinoside, in transgenic tobacco overexpressing the SoMYB1 gene is significantly increased.

[0068] In summary, the present invention has found for the first time that, compared with wild-type tobacco, the content of anthocyanin metabolites, especially cyanidin-3-O-rutinoside, in transgenic tobacco overexpressing the SoMYB1 gene is significantly increased. The SoMYB1 gene of the present invention, and its recombinant vector and recombinant bacteria can be used to increase the content of cyanidin-3-O-rutinoside in plants. The method for constructing a high-yield cyanidin-3-O-rutinoside transgenic plant provided by the present invention has broad application prospects in the directional production of high-yield cyanidin-3-O-rutinoside transgenic plants.

Claims

1. A SoMYB1 gene, characterized in that Its CDS sequence is shown in SEQ ID No.

1.

2. A recombinant vector, characterized in that: It includes the gene whose CDS sequence is shown in SEQ ID No.

1.

3. A recombinant bacterium, characterized in that: It comprises the recombinant vector according to claim 2.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria is recombinant Agrobacterium.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant Agrobacterium is a recombinant Agrobacterium tumefaciens.

6. A method for preparing transgenic tobacco with high yield of cyanidin-3-O-rutinoside, characterized in that: The method comprises transferring the SoMYB1 gene described in claim 1 into tobacco to obtain tobacco expressing the SoMYB1 protein.

7. The method according to claim 6, characterized in that The method for transferring into tobacco is one of the following: Agrobacterium method, gene gun method, electrotransformation method, PEG-mediated method, liposome method, and calcium phosphate-DNA coprecipitation method.

8. Use of the SoMYB1 gene according to claim 1, the recombinant vector according to claim 2 or the recombinant bacteria according to any one of claims 3 to 5 in preparing transgenic tobacco with high yield of cyanidin-3-O-rutinoside.

Images