Soybean MYB transcription factor gene and application thereof in increasing isoflavone content

Abstract

The invention is suitable for the technical field of gene engineering, and provides a soybean MYB transcription factor gene and application thereof in increasing the content of isoflavone. The soybean MYB transcription factor gene is GmMYB3a, and the nucleotide sequence of the soybean MYB transcription factor gene is as shown in SEQ ID NO. 1. According to the invention, a pTF101-GmMYB3a overexpression vector and an RNAi-GmMYB3a interference vector are successfully constructed, and the constructed vectors are transferred into a receptor soybean variety Williams 82. Experimental results show that the soybean strain with overexpression of the GmMYB3a gene shows higher isoflavone content, while the soybean strain with interference of the GmMYB3a gene shows lower isoflavone content, and the positive regulation effect of the GmMYB3a in the isoflavone biosynthesis process is proved. The invention not only provides an innovative molecular breeding strategy for cultivating soybean strains with high isoflavone content, but also has remarkable application potential and market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a soybean MYB transcription factor gene and an application thereof in improving the isoflavone content. Background Art

[0002] Soybean (Glycine max) is one of the most important economic crops in the world, providing humans with abundant vegetable oils, proteins and other nutrients. In recent years, with the publication of research results in the fields of diet and health, epidemiology, etc., soy isoflavones, a naturally occurring phytoestrogen, as an important member of flavonoid compounds, have attracted much attention for their physiological functions. Soy isoflavones show a wide range of physiological activities in estrogen regulation, anti-cancer, anti-oxidation and treatment of cardiovascular diseases. It is worth noting that isoflavones are mainly found in leguminous plants, and among edible leguminous plants, soybeans have the highest isoflavone content, which is almost 100 times that of other leguminous plants. Therefore, exploring and identifying genes related to isoflavones is of great significance to improving soybean varieties.

[0003] Soy isoflavones are a class of secondary metabolites synthesized by a branch of the phenylpropanoid / flavonoid metabolic pathway. The phenylalanine metabolic pathway has been well studied in the past few decades. It is initiated by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and coumarate coenzyme A ligase (4CL), and is converted and generated by a series of enzymatic reactions. In the phenylalanine metabolic pathway, the expression of catalytic enzyme genes is regulated by transcription factors. Among them, MYB transcription factors are one of the largest transcription factor families in the plant kingdom, and play an important regulatory role in multiple processes such as participating in plant growth and development, regulating the accumulation of flavonoid compounds and the synthesis of isoflavones. The study found that Zhu Ying et al. cloned an R2R3-MYB transcription factor related to the synthesis of soybean isoflavones. Quantitative analysis showed that GmMYB184 had the same expression pattern as IFS2. In addition, it was found that GmMYB184 could promote the expression of CHS8 and IFS2 gene promoters, and the activity was increased by 7 times and 5 times, respectively. The direct role of GmMYB184 in regulating isoflavone synthesis was further confirmed by the genetic transformation system mediated by Agrobacterium rhizogenes. After silencing GmMYB184, the isoflavone content in soybean hairy roots decreased significantly. Similarly, Pyrus betulifolia PbMYB12b and Malus domestica MdMYB22 can positively regulate the biosynthesis of flavonols. Soybean GmMYB176 regulates isoflavone biosynthesis by activating the expression of CHS genes. These research results show that MYB transcription factors play an important role in the regulatory mechanism of phenylpropanoid metabolic pathways. In breeding practice, the accumulation of isoflavones in soybean can be regulated by regulating the expression of MYB transcription factors, thereby cultivating excellent materials. However, due to the limited genetic variation related to soybean isoflavone content in germplasm resources and mutagenic materials, it is difficult to obtain soybean materials with high isoflavone content through conventional breeding methods, resulting in great challenges in the cultivation of high-isoflavone soybean varieties and slow research progress. So far, there is no report on the creation of high isoflavone transgenic soybean materials using soybean GmMYB3a gene. In view of this, the present invention proposes a soybean MYB transcription factor gene and its application in increasing the isoflavone content. Summary of the invention

[0004] The purpose of the present invention is to provide a soybean MYB transcription factor gene and its application in increasing the isoflavone content, aiming to solve the problems raised in the above background technology.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A soybean MYB transcription factor gene, the transcription factor gene is GmMYB3a, the nucleotide sequence thereof is shown as SEQ ID NO.1, and the amino acid sequence encoded by the gene is shown as SEQ ID NO.2.

[0007] An application of the soybean MYB transcription factor gene described above in increasing the isoflavone content of soybean strains.

[0008] Furthermore, the isoflavone content of soybean lines was increased by increasing the expression of the GmMYB3a gene in soybeans.

[0009] An overexpression vector pTF101-GmMYB3a containing the soybean MYB transcription factor gene as described above.

[0010] An overexpression strain containing the overexpression vector pTF101-GmMYB3a as described above.

[0011] A method for obtaining the above-mentioned overexpression strain comprises the following steps:

[0012] The soybean MYB transcription factor gene overexpression vector pTF101-GmMYB3a was transformed into the recipient soybean variety Williams 82 using Agrobacterium-mediated genetic transformation.

[0013] An application of the soybean MYB transcription factor gene described above in preparing transgenic plants.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a soybean MYB transcription factor gene and its application in improving the isoflavone content of soybean strains. Through molecular cloning technology, the present invention successfully cloned the key transcription factor gene GmMYB3a that regulates the phenylpropanoid metabolic pathway, and constructed a pTF101-GmMYB3a overexpression vector and an RNAi-GmMYB3a interference vector. The above-constructed vector was transferred into the recipient soybean variety Williams 82 using an Agrobacterium-mediated genetic transformation method. The experimental results showed that the expression level of the GmMYB3a gene in the overexpression strain was significantly higher than that in the control group, while the expression level of the GmMYB3a gene in the interference strain was significantly lower than that in the control group. In addition, the soybean strain overexpressing the GmMYB3a gene showed a higher isoflavone content, while the soybean strain interfering with the GmMYB3a gene had a reduced isoflavone content, confirming the positive regulatory effect of GmMYB3a in the isoflavone biosynthesis process. The present invention not only provides an innovative molecular breeding strategy for cultivating soybean strains with high isoflavone content, but also has significant application potential and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the electrophoresis result of PCR amplification of GmMYB3a; M: DL2000 marker; +: positive control; -: negative control; 1-6: samples.

[0017] Figure 2 Schematic diagram of pTF101-GmMYB3a and RNAi-GmMYB3a recombinant vectors; A is a schematic diagram of pTF101-GmMYB3a recombinant vector, and B is a schematic diagram of RNAi-GmMYB3a recombinant vector.

[0018] Figure 3 This is the PCR result of Agrobacterium tumefaciens transformed with pTF101-GmMYB3a and RNAi-GmMYB3a recombinant plasmids; M in the figure: Marker; +: positive control; -: negative control; 1: overexpression vector pTF101-GmMYB3a; 2: interference vector RNAi-GmMYB3a.

[0019] Figure 4 The flowchart of genetic transformation of soybean cotyledon node for overexpressing and interfering with GmMYB3a gene in soybean; wherein A is a schematic diagram of sterilizing seeds, B is a schematic diagram of inoculating seeds with hilum downward on germination medium, C is a schematic diagram of placing cotyledons with inner surface downward on solid co-cultivation medium with sterilization paper attached to the surface, D is a schematic diagram of inserting explants into screening medium, E is a schematic diagram of transferring explants into bud elongation medium, and F is a schematic diagram of moving rooted plants into pots for cultivation.

[0020] Figure 5 These are the results of bar test strips testing of some transgenic plants; in the figure, 1: control; 2-4: OE-MYB3a-1, OE-MYB3a-2, OE-MYB3a-3; 5-7: RNAi-MYB3a-1, RNAi-MYB3a-2, RNAi-MYB3a-3.

[0021] Figure 6 This is a graph showing the results of real-time fluorescence quantitative PCR detection of the GmMYB3a gene in R7 embryos of the T3 transgenic line.

[0022] Figure 7 This is a graph showing the total content of isoflavones in mature grains of T3 generation overexpression lines, interference lines and control plants.

[0023] Figure 8 The graph shows the total content of isoflavones in mature grains of T4 generation overexpression lines, interference lines and control plants. DETAILED DESCRIPTION

[0024] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0025] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0026] Example 1: One embodiment of the present invention provides a soybean MYB transcription factor gene, wherein the transcription factor gene is GmMYB3a, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2.

[0027] The following are the specific steps to obtain the transcription factor gene GmMYB3a:

[0028] Step 1: 40 mg of immature embryos (R5 stage) of soybean Jilin 32 were used as samples to extract total RNA from immature embryos of soybean variety Jilin 32. The integrity of RNA was tested by 1% agarose gel electrophoresis. cDNA was synthesized according to The procedure was performed according to the instructions of First-Strand cDNA Synthesis SuperMix.

[0029] Step 2: Design primer sequences according to the CDS sequence of GmMYB3a in the NCBI database (accession number: XM_003556598):

[0030] GmMYB3a-F: 5'-ATGAGGAAGCCTAGTTGT-3' (as shown in SEQ ID NO. 3);

[0031] GmMYB3a-R: 5'-TTACTGAAACAGAAGAAG-3' (as shown in SEQ ID NO. 4);

[0032] Using soybean Jilin 32 immature embryo cDNA as a template, PCR amplification was performed according to the following reaction system and conditions. Reaction system: 25μL system, including 10×PCR Buffer 2.5μL, 2.5mM dNTP mix 2μL, 10μM GmMYB3a-F and GmMYB3a-R 1μL each, cDNA 1μL, Taq DNA Polymerase 0.5μL and deionized water 17μL. Reaction conditions: pre-denaturation 94℃5min; 94℃30sec, 55℃30sec, 72℃60sec, a total of 35 cycles; post-extension 72℃5min. Finally, the PCR product of the full-length CDS of GmMYB3a was obtained ( Figure 1 ).

[0033] Step 3: The PCR product was subjected to agarose gel electrophoresis, and the target fragment was recovered by gel cutting. The recovered fragment was connected to the intermediate vector pMD18-T (purchased from TaKaRa), and then the ligation product was transferred into Escherichia coli (DH5α) competent cells. Escherichia coli was screened and cultured on LB solid medium containing ampicillin, single clones were picked and tested by PCR, the positive single clone bacterial solution was sequenced, the correctly sequenced bacterial solution was preserved and the plasmid (pMD18-T-GmMYB3a) was extracted for later use.

[0034] Example 2: One example of the present invention provides an application of a soybean MYB transcription factor gene in increasing the isoflavone content of soybean strains.

[0035] 1. Construction of GmMYB3a gene overexpression and interference vectors and genetic transformation and expression analysis in soybean;

[0036] Step 1: Construction of GmMYB3a gene overexpression vector and interference vector;

[0037] (a) Construction of the plant overexpression vector pTF101-GmMYB3a of the GmMYB3a gene: Primer sequences with XbaI and SacI restriction endonuclease sites were designed based on the CDS sequence of GmMYB3a ( Figure 2 A), namely pTF101-GmMYB3a-F and pTF101-GmMYB3a-R; then, using the pMD18-T-GmMYB3a plasmid extracted and sequenced correctly in step 3 of Example 1 as a template, PCR amplification was performed to obtain an amplified product of the GmMYB3a sequence containing the restriction site.

[0038] The specific primer sequences are:

[0039] pTF101-GmMYB3a-F:

[0040] 5'-TGCTCTAGACAAAGGACATGGATCGGATAAAAGG-3' (as shown in SEQ ID NO. 5);

[0041] pTF101-GmMYB3a-R:

[0042] 5'-CGAGCTCTTATTCAACCCTACCAATTCCCATCC-3' (as shown in SEQ ID NO. 6);

[0043] (b) Construction of RNAi-GmMYB3a vector for interfering GmMYB3a gene: The 1-300 bp sequence of GmMYB3a ORF sequence was selected as the cloned fragment, and the XbaI / HindIII and XhoI / KpnI restriction sites were added to both ends of the cloned fragment and its reverse sequence, and the two sequences were connected to the two sides of the PDK intron of the intermediate vector PKANNIBAL by homologous recombination ( Figure 2 (middle B).

[0044] The amplified product was connected to the vector fragment with restriction sites using a seamless cloning kit to construct a recombinant plasmid. Subsequently, the ligation product was transformed into Escherichia coli (DH5α) competent cells, and Escherichia coli was screened and cultured on LB solid medium containing kanamycin. Single clones were picked and tested by bacterial liquid PCR, and two 672bp bands were found ( Figure 3 ), and the positive monoclonal bacterial solution was sequenced. When the sequencing showed that the GmMYB3a gene overexpression vector and the interference vector were successfully constructed, the obtained pTF101-GmMYB3a and RNAi-GmMYB3a plasmids were transferred into the Agrobacterium competent EHA101 for subsequent bacterial solution preparation.

[0045] Step 2: Genetic transformation of pTF101-GmMYB3a overexpression vector and RNAi-GmMYB3a interference vector in soybean;

[0046] Step 2.1: Agrobacterium-mediated cotyledonary node infection method;

[0047] Step 2.1.1: Seed sterilization;

[0048] Select mature Williams 82 seeds with smooth skin, full grains and no disease spots and place them in a petri dish. Place them in a fume hood desiccator and sterilize them with chlorine gas produced by 96 ml of sodium hypochlorite and 4 ml of concentrated hydrochloric acid ( Figure 4 Middle A), lasting 16-20h;

[0049] Step 2.1.2: Seed germination;

[0050] After sterilization, the seeds were inoculated with the hilum facing downward on the germination medium ( Figure 4 Middle B), dark culture for 16 h;

[0051] Step 2.1.3, bacterial solution preparation;

[0052] The EHA101 Agrobacterium strain carrying the target gene was streaked on solid YEP medium containing 25 mg / L rifampicin and 25 mg / L spectinomycin. After inverted culture at 28°C for 2-3 days, a single clone was picked and inoculated into 3 mL liquid medium containing 25 mg / L rifampicin and 25 mg / L spectinomycin, and cultured overnight at 28°C and 220 rpm. The bacterial solution was then transferred to 50 mL of fresh YEP liquid medium, cultured overnight at 28°C and 220 rpm, centrifuged at 5000 rpm for 10 min, and resuspended in liquid LCCM medium to an OD of 600 =0.6;

[0053] Step 2.1.4, infection and co-cultivation;

[0054] Select beans that are not contaminated and grow well, carefully peel off the seed coat with tweezers, cut off the radicle and most of the hypocotyl and a pair of true leaves, cut a knife at the top of the embryo, put the cut beans in the infection solution, shake gently on the shaker, and infect for more than 2 hours. After the infection is completed, pour out the bacterial solution and place the cotyledons with the inner side facing down on the solid co-cultivation medium with sterilized paper on the surface ( Figure 4 Middle C), cultured in the dark for 4 days;

[0055] Step 2.1.5: Bud induction;

[0056] The explants were removed from the co-cultivation medium and placed on the recovery medium, and cultured at 24°C and 8 / 16 light for 14 days; after 14 days, the explants were removed and inserted into the screening medium ( Figure 4 Middle D), continued to culture under the same conditions under illumination for 14 days;

[0057] Step 2.1.6: bud elongation;

[0058] Select some well-growing explants, remove large and dead buds, transfer the explants to bud elongation medium, and culture at 24°C and 18 / 6 light intensity ( Figure 4 Middle E), subculture every two weeks;

[0059] Step 2.1.7: Rooting;

[0060] When the elongated regenerated buds reach 2-3 cm, they are treated for rooting and transferred to rooting induction medium. They are cultured until roots grow out and then transferred to pots with a soil-vermiculite ratio of 1:1 for culture ( Figure 4 Middle F).

[0061] Step 2.2: Detection of transformed plant progeny;

[0062] The transgenic plants obtained from the T0 generation were cultured for generations, and stable transgenic lines were obtained in the T3 and T4 generations, namely OE-MYB3a-1, OE-MYB3a-2, OE-MYB3a-3 (overexpression lines); RNAi-MYB3a-1, RNAi-MYB3a-2, RNAi-MYB3a-3 (interference lines). Each generation of plants was tested by bar test strips. The specific operation was as follows: fresh leaves were taken, ground into homogenate, and after centrifugation, the leaf homogenate was taken into a new sterile 1.5mL centrifuge tube, and the test strip (Liberty Link strip) was taken out and inserted into the homogenate for testing. The results were observed after 5 minutes. If two bands were displayed, it was a transgenic plant, and if there was only one indicator band, it was a wild-type non-transgenic plant. Figure 5 Some bar test results were displayed, and a total of 6 transgenic strains were detected, including 3 overexpression strains and 3 interference strains.

[0063] Step 3: Analysis of transcriptional levels of target genes in transgenic plants; specifically including:

[0064] Step 3.1: Total RNA was extracted from R7 embryos of T3 and T4 soybean varieties Williams 82 (W82) and transgenic lines OE-MYB3a and RNAi-MYB3a, and the extracted total RNA was reverse transcribed into cDNA using the same method as step 1 in Example 1.

[0065] Step 3.2: Real Time PCR was used to detect the expression level of the target gene in each strain according to the instruction manual of TransStart Tip Green qPCR SuperMix (purchased from Quanshijin) and using Agilent Technologies Stratagene Mx305P amplification instrument.

[0066] Among them, soybean β-tubulin was used as the internal reference gene, and the primer sequences were:

[0067] Tubulin-F: 5'-GGAAGGCTTTCTTGCATTGGTA-3' (as shown in SEQ ID NO. 7);

[0068] Tubulin-R: 5'-AGTGGCATCCTGGTACTGCA-3' (as shown in SEQ ID NO. 8);

[0069] Design specific primers for GmMYB3a gene:

[0070] Forward primer (F): 5'-TGCTCTAGACAAAGGACATGGATCGGATAA-3' (as shown in SEQ ID NO.9);

[0071] Reverse primer (R): 5'-CGAGCTCTTATTCAACCCTACCAATTCCCAT-3' (as shown in SEQ ID NO. 10);

[0072] The PCR reaction system is as follows:

[0073] Table 1 PCR reaction system

[0074]

[0075] The PCR reaction conditions were:

[0076]

[0077] The results showed that the expression level of the GmMYB3a gene in the T4 transgenic soybean lines was significantly different from that in the control plants (CK). Specifically, the expression level of the GmMYB3a gene in the overexpression lines was significantly higher than that in the control group, while the expression level of the GmMYB3a gene in the interference lines was significantly lower than that in the control group ( Figure 6 ). This phenomenon indicates that the transcriptional activity of the GmMYB3a gene is effectively regulated in the T4 generation of transgenic soybean lines, and the gene exhibits stable genetic characteristics in the transgenic soybean lines.

[0078] 2. Effects of GmMYB3a gene overexpression and interference on isoflavone content in mature soybean seeds;

[0079] Step 1: Extraction of isoflavones from mature soybean seeds;

[0080] Grind the soybean seeds into soybean powder with a grinder, pass through a 60-mesh sieve, accurately weigh 0.1 g of soybean powder, and put it into an explosion-proof glass tube. Then, add 10 ml of 80% methanol extract to each tube, heat it in an 80°C water bath for 14 h, centrifuge it at 12000 rpm for 15 min, filter the supernatant with a 0.45 μm filter membrane to obtain the final pretreatment solution, and store it at 4°C.

[0081] Step 2: Determination of isoflavone content by HPLC;

[0082] The isoflavone content was determined using a Japanese Tsushima high performance liquid chromatograph (LC-20A). The chromatographic system included: DGU-20A3 degasser, 2 LC-20AT solvent array detectors, UV detector SPD-20AV UV detector, CTO-10AS column oven, CBM-20A system controller, LC-Solution workstation and 7725 manual injector. The chromatographic column was Phenomenex C18 (150 mm × 4.6 mm, 5.0 μm); the mobile phase was methanol: water = 5:95 (v / v); the flow rate was 1 mL min -1 ; Detection wavelength: 254nm; Column temperature: 40℃; Injection volume: 10μL; Analysis time: 35-45min per sample.

[0083] All samples were measured three times in parallel, and the average value of the peak area was calculated. Qualitative analysis was performed based on the retention time, quantitative analysis was performed based on the peak area, and the content of each isoflavone component in the sample was calculated based on the standard curve of the standard.

[0084] The results showed that in the T3 generation, the total isoflavone content of the control plant (W82) was 3407.30 μg / g. The total isoflavone content of the overexpression strain OE-MYB3a-1 (P3-1) reached 5994.83 μg / g, an increase of 75.93% over the control plant; the total isoflavone content of the OE-MYB3a-2 (P3-2) strain was 5753.42 μg / g, an increase of 68.86%; the total isoflavone content of the OE-MYB3a-3 (P3-3) strain was 6291.82 μg / g, an increase of 84.66%. In contrast, the total isoflavone content of the interference strain RNAi-MYB3a-1 (R3-1) was 2415.79 μg / g, a decrease of 29.1%; the total isoflavone content of the RNAi-MYB3a-2 (R3-2) strain was 2102.99 μg / g, a decrease of 38.28%; the total isoflavone content of the RNAi-MYB3a-3 (R3-3) strain was 2575.66 μg / g, a decrease of 24.41% ( Figure 7 ).

[0085] In the T4 generation, the total isoflavone content of the control plant (W82) was 4124.16 μg / g. The total isoflavone content of the overexpression strain OE-MYB3a-1 (P3-1) reached 5566.47 μg / g, an increase of 34.97%; the total isoflavone content of the OE-MYB3a-2 (P3-2) strain was 5783.36 μg / g, an increase of 40.23%; the total isoflavone content of the OE-MYB3a-3 (P3-3) strain was 5499.09 μg / g, an increase of 33.34%. The total isoflavone content of the interference strain RNAi-MYB3a-1 (R3-1) was 3144.98 μg / g, a decrease of 23.74%; the total isoflavone content of the RNAi-MYB3a-2 (R3-2) strain was 2997.05 μg / g, a decrease of 27.33%; the total isoflavone content of the RNAi-MYB3a-3 (R3-3) strain was 2962.95 μg / g, a decrease of 28.16% ( Figure 8 ).

[0086] In the study of T3 and T4 generation lines, this study observed that soybean lines overexpressing the GmMYB3a gene significantly increased isoflavone content compared with control plants. In contrast, lines with interference with the GmMYB3a gene significantly reduced isoflavone content compared with control plants. These results indicate that the GmMYB3a gene plays a key role in regulating the biosynthesis of isoflavones in soybeans and has significant application potential. Therefore, the regulatory mechanism of the GmMYB3a gene has important scientific and commercial value for increasing soybean isoflavone production.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A soybean MYB transcription factor gene, characterized in that The transcription factor gene is GmMYB3a, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.

2.

2. Use of the soybean MYB transcription factor gene according to claim 1 in increasing the isoflavone content of soybean strains.

3. The use according to claim 2, characterized in that: Increasing the isoflavone content of soybean lines by increasing the expression of the GmMYB3a gene in soybean. 4 . An overexpression vector pTF101-GmMYB3a containing the soybean MYB transcription factor gene as claimed in claim 1 .

5. An overexpression strain containing the overexpression vector pTF101-GmMYB3a according to claim 4.

6. A method for obtaining the overexpression strain according to claim 5, characterized in that: The following steps are involved: The soybean MYB transcription factor gene overexpression vector pTF101-GmMYB3a was transformed into the recipient soybean variety Williams 82 using Agrobacterium-mediated genetic transformation.

7. Use of the soybean MYB transcription factor gene according to claim 1 in preparing transgenic plants.

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