Novel MYB transcription factor for improving content of anthocyanin in plant

Abstract

The invention belongs to the technical field of transgenic biosynthesis, and particularly relates to a novel MYB transcription factor for increasing the content of anthocyanin in plants. According to the invention, the SrMYB1 gene is separated from syringa reticulata, the SrMYB1 gene is subjected to genetic engineering technology, an expression vector is constructed, and transgenic tobacco over-expressing the SrMYB1 gene is obtained by using an agrobacterium-mediated infection method. Anthocyanin is remarkably accumulated in different tissues such as roots, stems, leaves, flowers, fruits and seeds of the transgenic tobacco overexpressed with the SrMYB1 gene, and the transgenic tobacco is purple; in leaves of the transgenic tobacco, the total anthocyanin content is about 12.8 times that of wild tobacco. It is shown that the SrMYB1 gene separated from syringa reticulata can be used for increasing the content of anthocyanin in tobacco, more choices are provided for biosynthesis of anthocyanin, and the SrMYB1 gene has a good application prospect in biosynthesis of anthocyanin.

Description

Technical Field

[0001] The invention belongs to the technical field of transgenic biosynthesis, and in particular relates to a novel MYB transcription factor for increasing the content of anthocyanins in plants. Background Art

[0002] Anthocyanins are a class of water-soluble pigments widely found in plants. They belong to the flavonoid class of compounds and are also the main coloring substances of plants. Anthocyanins in plants also have important functions such as anti-tumor, anti-inflammatory, and antioxidant in animals and humans. At present, anthocyanins have attracted widespread attention as a new type of medical resource.

[0003] The anthocyanin biosynthesis pathway has been widely studied in plants. Under the regulation of different genes, different types of anthocyanins are finally synthesized through a series of enzymatic reactions. There are two main types of genes involved in the biosynthesis of anthocyanins in plants. One type is structural genes, which can encode and synthesize various enzymes, and the other type is regulatory factors, which regulate the expression of structural genes. The regulatory factors that regulate anthocyanin biosynthesis mainly include three categories, MYB transcription factors, bHLH transcription factors, and WD40 transcription factors. The three mainly regulate the expression of structural genes by forming MBW complexes. MYB transcription factors are the main determinants in the MBW complex. Studies have found that MYB transcription factors determine the types and contents of anthocyanins by regulating the expression of structural genes in the anthocyanin biosynthesis pathway, ultimately affecting the color of different tissues of the plant.

[0004] The structural genes and regulatory genes that regulate anthocyanin synthesis have been verified in many plants, such as Arabidopsis, potato, wolfberry, grape, tea tree, etc. At present, the research on anthocyanin biosynthesis-related genes in cloves at home and abroad mainly focuses on the cloning of structural genes and transcriptional expression analysis, lacking the functional analysis of transcription factors that regulate anthocyanin synthesis, and it is impossible to determine which MYB transcription factors can be used to regulate anthocyanin biosynthesis through theoretical guidance, thereby increasing the content of anthocyanins in plants.

[0005] Due to its biological characteristics such as easy tissue manipulation, strong regeneration ability and large growth, tobacco has long been used as a research and development object and model organism for genetic engineering to synthesize and extract medicinal substances. Since 2008, using genetic engineering technology, tobacco has been used as a target to obtain drugs such as tumor cell surface proteins, ZMapp, serum protein activator TPA, cancer immune substances, and artemisinic acid, which are used to treat non-Hodgkin's lymphoma, Ebola virus infection, heart disease, cancer, malaria and other diseases.

[0006] There are no reports on the use of MYB transcription factors of Lilacinus officinalis to create transgenic tobacco rich in anthocyanins. In existing research results, people cannot use theoretical guidance to determine which MYB transcription factors in Lilacinus officinalis are involved in regulating the biosynthesis of anthocyanins, and it is difficult to use new regulatory genes to increase the content of anthocyanins. Therefore, exploring new regulatory genes to increase the content of anthocyanins is still a problem that needs to be solved in this field. Summary of the invention

[0007] The invention provides a SrMYB1 gene separated from Tobacco strychnifolia, aiming at increasing the anthocyanin content in tobacco.

[0008] The present invention provides a SrMYB1 gene, and the CDS sequence of the SrMYB1 gene is shown as SEQ ID No.1.

[0009] The present invention provides a recombinant plasmid, which comprises the CDS sequence of the SrMYB1 gene mentioned above.

[0010] Preferably, the recombinant plasmid is a plasmid obtained by inserting the CDS sequence of the SrMYB1 gene described above into a vector; the vector is pDONR207 and pJAM1502.

[0011] The present invention provides a recombinant bacterium, wherein the recombinant bacterium contains any of the recombinant plasmids described above.

[0012] Preferably, the recombinant bacterium is a recombinant Agrobacterium.

[0013] Preferably, the recombinant Agrobacterium is recombinant Agrobacterium tumefaciens.

[0014] The present invention provides a method for preparing a transgenic plant with high anthocyanin yield, which comprises transferring the above-mentioned SrMYB1 gene into a plant to obtain a plant expressing the SrMYB1 protein.

[0015] Preferably, the method of transferring the gene 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.

[0016] Preferably, the method of transferring the gene into plants is Agrobacterium tumefaciens.

[0017] Preferably, the plant is tobacco.

[0018] The present invention provides use of the above-mentioned SrMYB1 gene, any one of the above-mentioned recombinant plasmids, and any one of the above-mentioned recombinant bacteria in preparing a transgenic plant with high anthocyanin production.

[0019] Preferably, the transgenic plant is transgenic tobacco.

[0020] The present invention separates the SrMYB1 gene from the lilac, constructs an expression vector with the SrMYB1 gene through genetic engineering technology, and obtains transgenic tobacco overexpressing the SrMYB1 gene by using an Agrobacterium-mediated infection method. The transgenic tobacco overexpressing the SrMYB1 gene of the present invention has significantly accumulated anthocyanins in different tissues such as roots, stems, leaves, flowers, fruits, and seeds, and is purple; the total anthocyanin content in the leaves of the transgenic tobacco of the present invention is about 12.8 times that of wild-type tobacco. It shows that the SrMYB1 gene isolated from the lilac of the present invention can be used to increase the anthocyanin content in tobacco, provides more options for biosynthesis of anthocyanins, and has good application prospects in biosynthesis of anthocyanins.

[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 The agarose gel electrophoresis diagram of the SrMYB1 gene amplification product; wherein, lane M is the DNA marker D2000; and lanes 1-4 are the positive amplification products of the SrMYB1 gene.

[0024] Figure 2 The agarose gel electrophoresis diagram of the SrMYB1 gene connected to the pLB vector for screening bacteria; lane M is the D2000 DNA marker; lanes 1-5 are positive bacteria.

[0025] Figure 3 The agarose gel electrophoresis diagram of the amplification products of SrMYB1-attB1-F and SrMYB1-attB2-R primers; wherein, lane M is the D2000 DNA marker; lanes 1-6 are positive amplification products.

[0026] Figure 4 The figure is an agarose gel electrophoresis diagram of the constructed SrMYB1 expression vector; wherein, lane M is the DNA marker of D2000; lane 1 is the Bp reaction positive bacteria; lane 2 is the LR reaction positive bacteria; lane 3 is the Bp reaction positive bacteria plasmid; lane 4 is the LR reaction positive bacteria plasmid.

[0027] Figure 5The figure is an agarose gel electrophoresis diagram of SrMYB1 Agrobacterium transformation screening bacteria; wherein, lane M is the DNA marker of D2000; and lanes 1-10 are positive bacteria.

[0028] Figure 6 The figure shows the PCR identification results of the SrMYB1 overexpressing transgenic tobacco lines; lane M is the DNA marker of D2000; lanes 1-4 are positive amplification products.

[0029] Figure 7 Photos of wild-type tobacco and transgenic tobacco overexpressing SrMYB1.

[0030] Figure 8 The figure shows the results of the determination of the total anthocyanin content in the leaves of the SrMYB1 overexpressing transgenic tobacco line; WT is wild-type tobacco, SrMYB1 is the transgenic tobacco line, and FW is fresh weight. DETAILED DESCRIPTION

[0031] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0032] Example 1 Cloning of SrMYB1 gene

[0033] Through the MYB transcription factor known to regulate anthocyanin synthesis, homologous comparison was performed in the transcriptome database of the lilacinous genus to find the sequence of the target gene SrMYB1.

[0034] The TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China) was used to extract RNA from the leaves of P. salsa according to the instructions. TM 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China) was used to synthesize cDNA according to the instructions.

[0035] Using cDNA as a template, primers SrMYB1-F and SrMYB1-R were designed according to the coding region of the SrMYB1 gene, and the primer sequences were 5'-TCAGTCGATACAACTTTTTGGGA-3' (SEQ ID No. 2); 5'-AACAGACAAATCAAAATGGGAAACG-3' (SEQ ID No. 3), respectively, for PCR amplification. The amplification reaction system is shown in Table 1 below:

[0036] Table 1 PCR amplification reaction system

[0037]

[0038] The PCR reaction program was: 94°C, 3 min; 94°C, 30 s, 58°C, 30 s, 72°C, 90 s, 35 cycles; 72°C, 5 min, stored at 4°C. The PCR products were detected by 2% agarose gel electrophoresis. The results were as follows: Figure 1 As shown, it proves that the amplification is successful.

[0039] The target fragment of SrMYB1 was recovered using the SanPrep column DNA gel recovery kit (Shanghai Shenggong Biotechnology Service Co., Ltd.) according to the operating procedures in the manual. After connecting the target fragment, positive bacteria were screened according to the operating procedures in the pLB zero background rapid cloning kit (TIANGEN, China), and the positive bacteria were sent to Shanghai Shenggong for sequencing. After successful sequencing, the positive bacterial plasmid was extracted using the rapid plasmid extraction kit (TIANGEN, China) according to the operating procedures in the manual for the next step of expression vector construction. The results are shown in the figure. Figure 2As shown, it proves that the plasmid of the target gene (SrMYB1 gene) is successfully connected. Through sequencing, the CDS sequence of the SrMYB1 gene is determined to be: (SEQ ID No. 1).

[0040] The CDS of this gene is 714 bp.

[0041] Example 2 Construction and transformation of the expression vector of SrMYB1 gene

[0042] 1. Construction of SrMYB1 expression vector

[0043] The Gateway Cloning kit (Invitrogen, USA) was used to design primers SrMYB1-attB1-F and SrMYB1-attB2-R according to the instruction manual. The sequence of the SrMYB1-attB1-F primer is: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTTAGGCACAGAAGA-3' (SEQ ID No. 4), and the sequence of the SrMYB1-attB2-R primer is: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATATGATGTTTTCAGA-3' (SEQ ID No. 5). The SrMYB1 plasmid was amplified using 2×PfuPCR Mix (TIANGEN, China) according to the instruction manual. The reaction system for amplifying the SrMYB1 plasmid is as shown in Table 2:

[0044] Table 2 Reaction system for amplifying SrMYB1 plasmid

[0045]

[0046] The PCR reaction program was: 94°C, 3 min; 94°C, 30 s, 58°C, 30 s, 72°C, 2 min, 30 cycles; 72°C, 5 min, 4°C storage. The PCR product was detected by 2% agarose gel electrophoresis. The results are shown in Figure 3 As shown, it proves that the amplification is successful.

[0047] The PCR product was recovered and the SrMYB1 expression vector was constructed according to the instructions of the Gateway Cloning kit (Invitrogen, USA).

[0048] (1) Construction of entry vector (Bp reaction): The CDS sequence of the SrMYB1 gene was ligated into the entry vector pDONR207 to obtain pDONR207-SrMYB1.

[0049] (2) Construction of expression vector (LR reaction): pDONR207-SrMYB1 was recombined with the expression vector pJAM1502 to obtain the expression vector pJAM1502-SrMYB1.

[0050] The results are as follows Figure 4 As shown, it was proved that the expression vector pJAM1502-SrMYB1 was successfully constructed. The successfully constructed expression vector pJAM1502-SrMYB1 was transformed into Escherichia coli DH5α competent cells, positive bacteria were picked, and the plasmid was extracted for the next step of Agrobacterium transformation.

[0051] 2. Transformation of SrMYB1 expression vector into Agrobacterium

[0052] The pJAM1502-SrMYB1 expression vector was transferred into the Agrobacterium tumefaciens strain GV3101 by freeze-thaw method. The specific method is as follows:

[0053] 1. Take GV3101 Agrobacterium competent cells and place them on ice to dissolve. The reaction system is as follows: Take 1μL pJAM1502-SrMYB1 expression vector and 100μL Agrobacterium competent cells respectively. After fully mixing, place them in an ice bath for 30 minutes, then freeze them in liquid nitrogen for 5 minutes, take them out and immediately place them in a 37℃ water bath for 5 minutes, and finally place them on ice for 5 minutes.

[0054] 2. In a clean bench, add 500 μL of YEP liquid culture medium (Agrobacterium culture medium) to the above reaction system, seal it, and culture it on a shaker at 220 rpm and 28°C for 2 h.

[0055] 3. In the clean bench, take 100 μL of the shaken bacterial solution and spread it on the YEP solid culture medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it upside down at 28°C in the dark for two days.

[0056] 4. PCR detection was performed on the grown colonies using primers SrMYB1-attB1-F and SrMYB1-attB2-R. The results are as follows Figure 5 As shown, it was proved that the SrMYB1 expression vector was successfully transformed into Agrobacterium.

[0057] 3. SrMYB1 gene transformation into tobacco

[0058] 1. Conversion method

[0059] The pJAM1502-SrMYB1 expression vector was transformed into the tobacco genome using the Agrobacterium-mediated transformation method. The specific method is as follows:

[0060] (1) After culturing tobacco (Nicotiana tabacum L.) aseptic tissue culture seedlings for 2-3 weeks, the leaves were cut into a shape of about 1 cm in length and 1 cm in width. The cut leaves were placed in an Agrobacterium solution containing the pJAM1502-SrMYB1 expression vector and infected for 10 minutes. The concentration of Agrobacterium was about 0.6 (OD600).

[0061] (2) After the bacterial solution on the leaves was dried with sterile absorbent paper, the leaves were placed on a basal medium and cultured at 28°C in the dark for 2 days. The leaves were then transferred to a basal medium containing 300 mg / L cephalosporin and 50 mg / L kanamycin (Diamond, Shanghai, China) for resistance screening.

[0062] (3) When the leaf-differentiated seedlings grew to about 1.5 cm, the seedlings were transferred to MS medium containing 300 mg / L cephalosporin and 50 mg / L kanamycin and cultured for 20 days at 25°C to obtain SrMYB1 gene-transformed tobacco.

[0063] 2. Identification and detection of transgenic tobacco overexpressing the SrMYB1 gene

[0064] (1) PCR identification

[0065] Experimental method: Use specific primers SrMYB1-attB1-F and SrMYB1-attB2-R to amplify the DNA of transformed tobacco and identify transgenic tobacco. The amplification reaction system of the SrMYB1 gene in tobacco is shown in Table 3:

[0066] Table 3 Amplification reaction system of SrMYB1 gene in tobacco

[0067]

[0068] The PCR reaction program was: 94°C, 3 min; 94°C, 30 s, 58°C, 30 s, 72°C, 90 s, 35 cycles; 72°C, 5 min, stored at 4°C. The PCR products were detected by 2% agarose gel electrophoresis.

[0069] Experimental results: Through PCR detection, a total of 4 transgenic tobacco lines overexpressing SrMYB1 were screened, such as Figure 6 shown.

[0070] (2) Phenotypic observation and identification

[0071] like Figure 7 As shown. Phenotypic observation of transgenic tobacco strains revealed that, compared with wild-type tobacco (Nicotiana tabacum L.), different tissues of transgenic tobacco significantly accumulated anthocyanins. Wild-type tobacco roots are white, stems, leaves, fruits, and seeds are all green, and flowers are light pink; compared with wild-type tobacco, the roots, stems, leaves, flowers, fruits, and seeds of transgenic tobacco significantly accumulated anthocyanins. This proves that the transformation method of this embodiment successfully obtained a transgenic tobacco strain that accumulated anthocyanins.

[0072] (3) Total anthocyanin detection

[0073] Total anthocyanin content detection method: Take wild-type tobacco (Nicotiana tabacum L.) leaves and transgenic tobacco leaves obtained in this example, accurately weigh 0.1g tobacco leaves, place in a 50ml brown volumetric flask, add 25ml extract (concentrated HCl: 80% methanol = 1: 1000, V / V), plug and weigh, weigh again after ultrasonic 1h, make up the insufficient amount with extract, shake well, and obtain the test solution of wild-type tobacco leaves and the test solution of transgenic tobacco leaves respectively. Prepare 2 portions of the test solution. One portion is diluted with potassium chloride buffer solution (0.025mol / L, pH 1.0), and the other portion is diluted with sodium acetate buffer solution (0.4mol / L, pH 4.5) (test solution: buffer solution = 1: 4, v / v), and the absorbance of the sample is measured at 530nm and 700nm within 20-50min, respectively, to calculate the anthocyanin content.

[0074] Test results: Figure 8 The total anthocyanin content was detected and it was found that the total anthocyanin content in the leaves of the transgenic tobacco strains overexpressing the SrMYB1 gene was significantly increased, about 12.8 times the total anthocyanin content of the wild-type tobacco leaves (Nicotiana tabacumL.).

[0075] Through the above examples, it can be seen that the present invention isolates the SrMYB1 gene from the lilac, and the SrMYB1 gene is subjected to genetic engineering technology to construct an expression vector, and the transgenic tobacco overexpressing the SrMYB1 gene is obtained by using the Agrobacterium-mediated infection method. The roots, stems, leaves, flowers, fruits, seeds and other different tissues of the transgenic tobacco of the present invention all significantly accumulate anthocyanins and are purple; the total anthocyanin content in the leaves of the transgenic tobacco of the present invention is about 12.8 times that of the wild-type tobacco. It shows that the SrMYB1 gene isolated from the lilac of the present invention can be used to increase the anthocyanin content in tobacco, providing more options for the biosynthesis of anthocyanins, and has a good application prospect in the biosynthesis of anthocyanins.

Claims

1. A SrMYB1 gene, characterized in that: The CDS sequence of the SrMYB1 gene is shown in SEQ ID No.

1.

2. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the CDS sequence of the SrMYB1 gene according to claim 1.

3. A recombinant bacterium, characterized in that: The recombinant bacteria contain the recombinant plasmid according to claim 2 or 3.

4. The recombinant bacterium according to claim 3, characterized in that: The recombinant bacteria are 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 a transgenic plant with high anthocyanin production, characterized in that: The invention transfers the SrMYB1 gene described in claim 1 into plants to obtain plants expressing SrMYB1 protein.

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

8. The method according to claim 7 or 8, characterized in that: The plant is tobacco.

9. Use of the SrMYB1 gene according to claim 1, the recombinant plasmid according to claim 2, and the recombinant bacteria according to any one of claims 3 to 5 in preparing a transgenic plant with high anthocyanin production.

10. The use according to claim 9, characterized in that: The transgenic plant is transgenic tobacco.

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