Application of soybean flavanone-3-hydroxylase and flavone synthase genes

Abstract

The invention discloses an application of a soybean flavanone-3-hydroxylase gene and a soybean flavanone-3-hydroxylase gene and a soybean flavanone-3-hydroxylase gene. The nucleotide sequences of the soybean GmF3H1, the soybean GmF3H2 and the soybean GmFNSII-1 are as follows: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively. The CRISPR / Cas9 is used for simultaneously knocking out GmF3H1, GmF3H2 and GmFNSII-1 in the soybean, so that the expression level of the CRISPR / Cas9 in the soybean can be obviously reduced. Compared with a wild type, the hundred-grain weight, the branching number and the protein content of the three-gene mutant material are also stably increased in multiple generations. Therefore, the soybean flavanone-3-hydroxylase genes GmF3H1 and GmF3H2 and the flavone synthase gene GmFNSII-1 disclosed by the invention can be applied to the aspects of soybean hundred-grain weight, plant type improvement and high-protein variety quality improvement of soybean knockout strains through gene engineering regulation.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of soybean flavanone-3-hydroxylase and flavone synthase genes, and belongs to the field of genetic engineering, in particular to the application of increasing the branch number, hundred-grain weight and protein content of soybean after knocking out the flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 derived from soybean by using the CRISPR / Cas9 system. BACKGROUND

[0002] Soybean [Glycine max (L.) Merr.] is an important food and oil crop, and is one of the most important plant protein sources for humans. In addition to containing rich nutrients, soybean also contains many physiologically active substances, such as isoflavones. Isoflavones are important secondary metabolites produced in legume crops. They play an important role in anticancer, prevention of osteoporosis, improvement of blood lipids, antioxidant, prevention of cardiovascular disease, alleviation of menopausal symptoms in women, improvement of immunity, antiviral, and beauty and skin care; they also have important functions in plant disease resistance, promotion of nodulation in legume crops, and participation in plant-microbe interactions. Therefore, it is of great significance to increase the content of isoflavones in soybean. The synthesis of isoflavones involves multiple enzymes, and the key step is the hydroxylation of flavanones (naringenin, glycyrrhizin, etc.) at the C-2 position catalyzed by IFS. IFS competes with flavone synthase (FNS) and flavanone-3-hydroxylase (F3H) for the common substrate naringenin. FNS catalyzes the synthesis of flavones from naringenin. F3H catalyzes the synthesis of flavonols, anthocyanins and proanthocyanidins from naringenin.

[0003] In the previous stage, a three-gene edited soybean material (three-hit material) of GmF3H1, GmF3H2 and GmFNSII-1 was obtained by using CRISPR / Cas9 gene editing technology. Compared with the control material, the three-hit material not only had a significantly increased isoflavone content, but also had a significantly increased branch number, hundred-grain weight and protein content, and the traits were stable in multiple generations. The current research shows that F3H and FNS not only participate in the synthesis of flavonols, anthocyanins and flavones, but also enhance the disease resistance and stress tolerance of plants. However, there is no report that F3H and FNS affect the branch number, hundred-grain weight and protein content. SUMMARY

[0004] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0005] The object of the application can be achieved by the following technical solutions.

[0006] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0007] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0008] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0009] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0010] The application discloses genetic engineering application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavone synthase gene GmFNSII-1 which affect branch number, hundred-grain weight and protein content in addition to affecting soybean isoflavone content.

[0011] Advantages

[0012] GmF3H1 and GmF3H2 belong to the soybean iron / ascorbate-dependent oxidoreductase family gene, encode flavanone-3-hydroxylase protein, and the gene is involved in flavonol and anthocyanin biosynthesis. GmFNSII-1 belongs to the soybean cytochrome P450 family gene, encodes flavone synthase protein, and the gene is involved in flavonoid biosynthesis. After knocking out GmF3H1, GmF3H2 and GmFNSII-1 simultaneously by using CRISPR / Cas9 system, the expression levels of GmF3H1, GmF3H2 and GmFNSII-1 in soybean are significantly reduced, the isoflavone content is greatly increased, and the branch number, hundred seed weight and protein content are also significantly increased. Therefore, GmF3H1, GmF3H2 and GmFNSII-1 can be used as targets for increasing the branch number, improving the hundred seed weight and protein content of soybean, and for improving the plant type and quality of high-protein varieties of soybean. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Sequence variation analysis of triple mutant GmF3H1, GmF3H2 and GmFNSII-1.

[0014] Figure 2 Expression levels of wild type and triple mutant GmF3H1, GmF3H2 and GmFNSII-1. *: P<0.05, **: P<0.01, ***: P<0.001. Two-tailed test, error bars represent ± standard deviation.

[0015] Figure 3 Protein and oil content analysis of wild type and triple mutant plants. (a) Protein content of wild type Jack and T3 generation triple mutant; (b) oil content of wild type Jack and T3 generation triple mutant; (c) protein content of wild type Jack and T4 generation triple mutant; (d) oil content of wild type Jack and T4 generation triple mutant. *: P<0.05, two-tailed test, error bars represent ± standard deviation.

[0016] Figure 4 Branch number and hundred seed weight analysis of wild type and triple mutant plants. (a) Phenotype of wild type and triple mutant plants, scale: 10 cm; (b) pod phenotype of wild type and triple mutant, scale: 1 cm; (c) seed phenotype of wild type and triple mutant, scale: 1 cm; (d) branch number of wild type and T3 generation triple mutant; (e) branch number of wild type and T4 generation triple mutant; (f) hundred seed weight of wild type and T3 generation triple mutant; (g) hundred seed weight of wild type and T4 generation triple mutant. WT: wild type; KO: triple mutant. *: P<0.05, **: P<0.01, ***: P<0.001. Two-tailed test, error bars represent ± standard deviation. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0019] Example: Genetic engineering applications of mutant soybean genes F3H1, F3H2, and FNSⅡ-1

[0020] 1) Construction of a three-stage carrier

[0021] To construct the pGmUbi-Cas9-4XsgR triple knockout vector targeting GmF3H1, GmF3H2, and GmFNSII-1, firstly, primers (GmF3H-F1 / R1 and GmFNSII-1-F1 / R1) with vector adapters and target sequences of GmF3H and GmFNSII-1 were synthesized, and primers for the vector sequence were also synthesized (the primers for the pU6-sgR vector were Sg1-F and Sg1-R). Using GmF3H-F1 / Sg1-R, Sg1-F / GmF3H-R1, GmFNSII-1-F1 / Sg1-R, and Sg1-F / GmFNSII-1-R1 as primer pairs, and pU6-sgR vector as template, PCR amplification was performed to obtain amplification products 1, 2, 3, and 4, respectively; 2) Amplification products 1 and 2, and 3 and 4 from 1) were fused and ligated using a bridging PCR method to obtain products pU6-sgR1 and pU6-sgR2; 3) The products from 2) were ligated to pClone007 using T4 ligase. T vector (catalog number: TSV-007BS) was used to obtain vectors T-pU6-sgR1 and T-pU6-sgR2; 4) PCR was performed using specific primers (pU6-F / R) with vectors T-pU6-sgR1 and T-pU6-sgR2 from 3) as templates to amplify modules GmU6pro-RNA1 and GmU6pro-RNA2.

[0022] Similarly, first 1) synthesis with carrier joint and with GmFNSII-1 target sequence of primer (GmFNSII-1-F2 / R2), while synthesis of vector sequence primer (pU3-sgR vector primer is RH-F, RH-R). Respectively with GmFNSII-1-F2 / RH-R and RH-F / GmFNSII-1-R2 as primer pair, both with pU3-sgR vector as template for PCR amplification, respectively, to obtain product 1 and 2; 2) by bridging PCR method to fuse the product in 1), to obtain product pU3-sgR; 3) using T4 ligase to connect the product in 2) to pClone007T vector, to obtain vector T-pU3-sgR; 3) using specific primer (pU3-F / R) with T-pU3-sgR vector as template for PCR, and then amplifying the module GmU3pro-RNA.

[0023] Using T4 ligase to connect the module GmU6pro-RNA1 and GmU3pro-RNA, module GmU6pro-RNA2 and GmU3pro-RNA to the double enzyme digested pGmUbi-Cas9 vector (NcoI and XbaI enzyme digestion), to obtain vector pGmUbi-Cas9-2XsgR1 and pGmUbi-Cas9-2XsgR2. In order to produce the final CRISPR / Cas9 multi gene editing vector, using specific primer (F / R) to PCR amplify the two tandem sgRNA modules in pGmUbi-Cas9-2XsgR1 vector, and the amplification product is recombined into another single enzyme pGmUbi-Cas9-2XsgR2 vector (BstEII enzyme digestion) using recombinase. Finally, the multi gene editing pGmUbi-Cas9-4XsgR vector assembled with 4 sgRNA was obtained, and table 1 was the primer used for constructing the vector.

[0024] Table 1 primer for constructing pGmUbi-Cas9-4XsgR vector

[0025]

[0026] Bold and lowercase letters represent linker sequences.

[0027] 2) Three burst material GmF3H1, GmF3H2 and GmFNSII-1 sequence variation analysis

[0028] The V3 stage wild type soybean Jack and three punctured material plants were taken, and the leaves were quickly frozen in liquid nitrogen and stored in a -80 refrigerator for three punctured material positive and homozygous identification. The total DNA was extracted by using a DNA extraction kit (purchased from Shanghai Puduo Biotechnology Co., Ltd.). The primer was designed by using Primer Premier 5, and the primer was synthesized by Genesee Biotechnology Co., Ltd. The primer sequence is shown in Table 2. In order to identify whether the three target genes GmF3H1, GmF3H2 and GmFNSII in the soybean plant are all mutated, the target site is amplified by using high fidelity enzyme (Novozyme Biotechnology) and target gene specific primer, and the PCR product is used for sequencing, and the sequencing is completed by Beijing Genesee Biotechnology Co., Ltd. The sequencing analysis result shows that three genes are all homozygous frame shift mutations. Figure 1 ).

[0029] Table 2 Three punctured material positive and homozygous identification primer

[0030]

[0031] 3) Wild type and three punctured material GmF3H1, GmF3H2 and GmFNSII-1 expression level

[0032] Jack was selected as the experimental variety, and the V6 stage leaf tissue of wild type and three punctured material plants was taken. The samples were quickly frozen in liquid nitrogen and stored at -80℃. The sample was ground with a mortar, and a 1.5 mL EP tube containing a lysis solution was added. After shaking, it was moved to a 1.5 mL EP tube, and total RNA was extracted by using an RNA extraction kit (Shanghai Puduo). The quality of total RNA was identified by gel electrophoresis, and the RNA content was determined by spectrophotometer. The total RNA of the above-mentioned tissue was used as a template, and reverse transcription was carried out to obtain cDNA. The GmF3H1, GmF3H2 and GmFNSII-1 fluorescence quantitative primer sequences were GmF3H1-Q, GmF3H2-Q and GmFNSII-Q (Table 3), respectively. The relative expression amount of the gene in each tissue was detected by real-time fluorescence quantitative PCR (Real-time RT-PCR), and the reagent for RT-PCR was ChamQ Universal SYBR qPCR Master Mix (Q711) of Novozyme. Soybean Tubulin (GenBank: AY907703.1) was used as an internal reference gene, and the primer sequence was Tublin-Q (Table 1). The expression amount of the target gene was calculated by using 2 -ΔΔCT The results show that compared with the control Jack, the expression amounts of F3H1, F3H2 and FNSII-1 genes in the three punctured material are significantly reduced. Figure 2

[0033] Table 3 Fluorescence quantitative related primer

[0034] ​

[0035] 4) Agronomic trait investigation of triple mutant materials

[0036] Wild type and triple mutant materials were planted in the greenhouse of Nanjing Agricultural University Baima Base. The growth conditions of plants were observed and recorded during the whole growth period. The agronomic traits such as initial flowering stage, full flowering stage, pod setting stage, grain filling stage, and maturity stage were investigated. The results showed that the flowering stage, pod setting stage, grain filling stage, and maturity stage of triple mutant materials were later than those of wild type.

[0037] Table 4 Investigation table of growth period of triple mutant materials and control Jack

[0038]

[0039] 5) Protein content, branch number, and hundred seed weight of triple mutant materials significantly increased

[0040] The determination results of protein and oil content of mature seeds showed that the protein content of triple mutant materials significantly increased. The protein content of control Jack was 38.40%, and the oil content of control Jack was 20.98%. The protein content of T3 generation of four strains of triple mutant materials was 40.45%-41.45%, which was significantly higher than that of control Jack (a). The oil content of T3 generation of triple mutant materials was 19.81%-20.68% (b), which had no significant difference with control. The protein content of T4 generation of four strains of triple mutant materials was 40.65%-40.92%, which was significantly higher than that of control Jack (c). The oil content of T4 generation of four strains of triple mutant materials was 19.35%-20.59% (d), which had no significant difference with control. Figure 3 Figure 3 Figure 3 Figure 3 Figure 3

[0041] Investigation and statistics of branch number of mature plants and hundred seed weight of seeds showed that the branch number and hundred seed weight of triple mutant materials were significantly increased compared with wild type (a, 4b, and 4c). In terms of branch number, the average branch number of control Jack was 3.33. In T3 generation of triple mutant materials, the average branch number of four strains was 4.67, 5.00, 5.29, and 6.00, respectively, which was significantly higher than that of control Jack (d). In T4 generation of triple mutant materials, the branch number of four strains was 4.50, 4.67, 5.00, and 4.75, respectively, which was significantly higher than that of control Jack (e). Figure 4 Figure 4 Figure 4

[0042] ​​​​​​​​In the aspect of 100-grain weight, the average 100-grain weight of the control Jack was 13.65 g. In the T3 generation lines of the three-hit material, the average 100-grain weights of the four lines were 20.10 g, 18.45 g, 19.30 g and 18.16 g respectively, which were significantly higher than that of the control Jack Figure 4 f) In the T4 generation lines, the average 100-grain weights of the four lines were 19.25 g, 19.31 g, 17.99 g and 19.52 g respectively, which were significantly higher than that of the control Jack Figure 4 g).

Claims

1. Application of soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavonoid synthase gene GmFNSII-1 in genetic engineering of soybean hundred kernel weight, branch number and protein content, wherein the coding region sequences of the soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavonoid synthase gene GmFNSII-1 are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

2. Use according to claim 1, characterized in that Knocking out the GmF3H1, GmF3H2 and GmFNSII-1 by using CRISPR / Cas9 system can improve the hundred kernel weight, branch number and protein content of soybean.

3. Application of a vector for simultaneously knocking out the soybean flavanone-3-hydroxylase genes GmF3H1, GmF3H2 and flavonoid synthase gene GmFNSII-1 in genetic engineering of soybean hundred kernel weight, branch number and protein content.

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