Application of GmPPT gene in increasing total fatty acid content of soybean

Abstract

The invention discloses application of a GmPPT gene in increasing the total fatty acid content of soybeans, and belongs to the technical field of plant breeding. The purpose of the invention is to increase the total fatty acid content of soybeans. The invention provides a method for increasing the total fatty acid content of soybeans. Gene shown in SEQ ID NO.7 is over-expressed in the soybeans to obtain transgenic soybeans. Excavation of an excellent gene for regulating and controlling the content of soybean fatty acid has important significance on genetic improvement of soybean oil.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically involving the application of the GmPPT gene in increasing the total fatty acid content of soybeans. Background Technology

[0002] Soybeans are one of the world's most important dual-purpose crops (food and cash crops) and a significant source of unsaturated fatty acids and protein for human consumption. In recent years, consumer demand for healthy oils has become increasingly urgent. Therefore, increasing the total fatty acid content and optimizing the fatty acid composition of soybean grains is one of the main goals of soybean genetic improvement breeding. Through long-term domestication and genetic improvement, the total fatty acid content of soybeans has generally shown a continuous upward trend, but the genetic basis regulating fatty acid content remains unclear. Soybean fatty acid content is a complex quantitative trait regulated by multiple genes, and its genetic improvement requires the coordinated action of multiple metabolic pathways. However, synthetic regulatory genes with significant utilization value are still rarely reported. Therefore, identifying superior genes regulating soybean fatty acid content is of great significance for the genetic improvement of soybean oil. Summary of the Invention

[0003] The purpose of this invention is to increase the total fatty acid content of soybeans.

[0004] This invention provides the application of the amino acid sequence shown in SEQ ID NO.8 in increasing the total fatty acid content of soybeans.

[0005] This invention provides an application of overexpressing the gene shown in SEQ ID NO.7 in increasing the total fatty acid content of soybeans.

[0006] This invention provides the application of a recombinant vector containing the gene shown in SEQ ID NO.7 in increasing the total fatty acid content of soybeans.

[0007] Further specifying, the originating vector for the recombinant vector is pCAMBIA3300.

[0008] This invention provides the application of a recombinant microorganism containing the gene shown in SEQ ID NO.7 in increasing the total fatty acid content of soybeans.

[0009] Furthermore, the originating microorganism for recombinant microorganisms is a prokaryotic microbial cell.

[0010] This invention provides a breeding method for increasing the total fatty acid content of soybeans. The specific steps of the breeding method are as follows: Step 1: Ligate the gene shown in SEQ ID NO.7 with the overexpression vector to obtain the recombinant vector; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 was transferred into sugar beets to obtain transgenic soybean hairy roots. After identification, positive transgenic soybean hairy roots were obtained.

[0011] To further specify, the primer pair used in step 1 to amplify the gene shown in SEQ ID NO.7 is SEQ ID NO.1 and SEQ ID NO.2.

[0012] Further specifying, the overexpression vector is pCAMBIA3300.

[0013] This invention provides a method for increasing the total fatty acid content of soybeans by overexpressing the gene shown in SEQ ID NO.7 in soybeans to obtain transgenic soybeans.

[0014] Beneficial effects: The total fatty acid content in the hairy roots of the control line was 0.691 mg / mL, while the total fatty acid content in the hairy roots of the GmPPT gene overexpressing line OE1 was 0.763 mg / mL and OE2 was 0.761 mg / mL. Moreover, the total fatty acid content of the GmPPT gene overexpressing lines (OE1 and OE2) was significantly higher than that of the control line. Attached Figure Description

[0015] Figure 1 The image shows the results of the basic characteristic analysis of the GmPPT gene. Figure 2 This is a phylogenetic tree diagram of the GmPPT system; Figure 3 Figure showing the expression pattern of the GmPPT gene in soybean tissues; Figure 4 Figure showing the classification and number of cis-regulatory elements in the GmPPT gene promoter; Figure 5 Figure showing the cloning results of the GmPPT gene; Note: M: DL2000; 1-4: PCR products; Figure 6 Figure showing the results of transforming DH5α Escherichia coli with a plant expression vector; Note: M: DL2000; 1-5: PCR products; Figure 7 This is a diagram showing the subcellular localization results of the GmPPT protein; Figure 8 Figure 1 shows the results of real-time PCR analysis of the GmPPT gene in different tissue sites. Figure 9 Figure 1 shows the PCR results of Agrobacterium rhizogenes culture containing the pCAMBIA3300-GmPPT recombinant plasmid; M: DL2000; 1-5: PCR products. Figure 10The figure shows the results of the GmPPT gene regulating the accumulation of total fatty acid content in soybean hairy roots. Detailed Implementation

[0016] Example 1. GmPPT Gene feature analysis Bioinformatics analysis of the coding sequence of the soybean plasmid phosphoenolpyruvate / phosphate translocator (PPT) gene revealed that valine (V) constitutes the largest proportion of the amino acid composition of the GmPPT protein, approximately 11.4%, while cysteine ​​(C) accounts for the smallest proportion, at 0.5%. The other five amino acids—pyrrolidone (O), serine (U), phenylalanine (B), threonine (Z), and phenylalanine (X)—do not participate in the biosynthesis of this protein. Figure 1 (A) The GmPPT protein has 34 positively charged residues (Arg+Lys) and 13 negatively charged residues (Asp+Glu), and its molecular structure is C. 2013 H 3119 N 513 O 530 S 14 It has a relative molecular mass of 43.43 kDa, a theoretical isoelectric point of 10.21, a total number of atoms of 6189, an aliphatic index of 96.72, an average hydrophilicity index of 0.387, and an instability coefficient of 49.27, classifying it as an unstable protein. Based on the protein's hydrophilicity... Figure 1 In sample B), GmPPT was found to have a predominance of hydrophilic amino acids along its entire polypeptide chain, exhibiting overall hydrophilicity and classifying it as a hydrophilic protein. GmPPT protein contains 40 potential phosphorylation sites, including 25 serine phosphorylation sites, 12 threonine phosphorylation sites, and 3 tyrosine phosphorylation sites. Figure 1 The C in the text). GmPPT protein does not have a signal peptide structure and belongs to the non-secretory protein category. Figure 1 The GmPPT protein, consisting of 396 amino acids, is composed of three structural types: α-helix (49.24%), random coil (41.16%), and extended chain (9.60%). Figure 1 (E in the text). The protein's tertiary structure is predominantly α-helical, which is highly consistent with the predicted secondary structure.

[0017] Phylogenetic analysis of the GmPPT gene showed that homologous genes exist in various plants. Comparative analysis of 39 homologous genes from soybean, maize, Arabidopsis, and other plants revealed that they can be divided into four subfamilies, with GmPPT showing the closest phylogenetic relationship to Arabidopsis. Figure 2 ).

[0018] Using the Ensembl Plants database GmPPT The relative expression of the gene in different parts of soybean was predicted, and the results showed that the gene was expressed at a higher level in seeds and leaves. Figure 3 ).

[0019] Selecting using the Phytozome database GmPPT Analysis of the promoter region of the 2000 bp upstream of the ATG gene using the Plant CARE online database revealed that the core elements AuxRR-core and TCA-element are involved in auxin and salicylic acid responses, respectively; WRE3 and WUN-motif are involved in gene regulation of wound response; and ERE and STRE are stress-related elements. AuxRR-core is involved in auxin-mediated cell elongation (e.g., stem elongation) and root development (e.g., lateral root formation). These elements, combined with upstream genes, jointly participate in… GmPPT The gene's regulated expression suggests that it is involved in plant growth and development as well as resistance to various stresses. Figure 4 ).

[0020] Target gene CDS cloning. The Phyzome V13 database was used to search for the soybean Williams 82 (Glycine maxWm82.a2.v1) gene sequence, followed by BLAST alignment to obtain the desired sequence. GmPPT Based on the CDS sequence information, gene cloning primers (primer 1) were designed using Primer 5.0 software. PCR was performed using cDNA as a template. The system configuration was 20 µL: GmPPT-3300-F 1 µL, GmPPT-3300-R 1 µL, template DNA 1 µL, KOD One™ PCR Master Mix (TOYOBO) 10 µL, and ddH2O 7 µL. The PCR program was as follows: pre-denaturation 98℃, 2 min; denaturation 98℃, 10 sec; annealing 60℃, 5 sec; extension 68℃, 10 min, for a total of 35 cycles, stored at 4℃. After the reaction, the PCR products were analyzed by agarose gel electrophoresis and the target fragment was purified by gel extraction. Figure 5 ).

[0021] Primer 1: Overexpression and subcellular localization primers GmPPT-3300-F: tcgagctccgtcgacaagcttATGCAGAGCGCGGCTTTC (SEQ ID NO.1); GmPPT-3300-R: gcccttgctcaccataagcttAGCTGTTTTTGGCTTTGCCTT (SEQ ID NO.2); Primer 2: Primers for real-time PCR GmPPT-qRT-F:ACCTGTTACCCACTCCG (SEQ ID NO.3); GmPPT-qRT-R: AATACGCTTCACCCTTG (SEQ ID NO.4); GmActin 4-F: GTTTCAAGCTCTTGCTCGTAATCA (SEQ ID NO. 5); GmActin 4-R: GTGTCAGCCATACTGTCCCCATTT (SEQ ID NO. 6); GmPPT Gene CDS sequence (SEQ ID NO.7): GmPPT gene amino acid sequence (SEQ ID NO.8): MQSAAFFTFSLPLRNPSPNYWRRPSLSLRLSAKHGNSNSDDVNSNGVSSTFFTRRSWTLPPSSSFKFRPLPPRAAESAVPESAPVENPLFKTLELGALFGLWYLFNIYFNIYNKQVLKAFHYPVTVTVVQFAVGTVLVAFMWGLNLYKRPKLSGAMLGAILPLAAVHTLGNLFTNMSLGKVAVSFTHTIKAMEPFFS VVLSAMFLGEFPTPWVVGSLVPIVGGVALASVTEASFNWAGFWSAMASNVTNQSRNVLSKKAMVNKEDSMDNITLFSIITVMSFFLLAPVAIFMEGVKFTPAYLQSAGVNVRQLYIRSLLAALCFHAYQQVSYMILQRVSPVTHSVGNCVKRVVVIVSVIFFQTPVSPVNAFGTAIALAGVFLYSRVKRIKAKPKTA.

[0022] Example 2. Vector construction and coliform transformation The linearized pCAMBIA3300 vector plasmid was homologously ligated to the target gene GmPPT using a homologous recombination kit. The ligation system consisted of 20 µL: plasmid 2 µL, GmPPT fragment 1 µL, ligase 2 µL, 5×CE Buffer 2 µL, and ddH2O 13 µL. The incubation program was 37℃ for 30 min. The ligation product was then transformed into DH5α competent cells, and single-clone cells were cultured. Positive clones were further identified using specific primers (primer 1). The cultured bacterial culture was used as a template for bacterial PCR identification (PCR program was the same as the GmPPT cloning program). After the reaction was complete, the results were detected by agarose gel electrophoresis, revealing a target fragment size of 1191 bp. Figure 6 The correctly identified bacterial cultures were further sequenced by Ribo Biotechnology Co., Ltd., and the sequences were aligned using DANMAN software. The correctly sequenced E. coli cultures were then amplified, and plasmid DNA was extracted from the amplified E. coli using the Plasmid Mini Kit I (Omega) kit.

[0023] Example 3. Subcellular localization and expression pattern analysis 1) Subcellular localization pCAMBIA3300- GmPPTThe recombinant plasmid was transformed into GV3101 (psoup-p19) competent cells to further clarify... GmPPT The functional location of a gene within the cell. pCAMBIA3300- GmPPT Agrobacterium infection was performed on young tobacco leaves, with pCAMBIA3300-GFP Agrobacterium serving as a control. The samples were observed under a laser confocal microscope. GmPPT The functional location of the gene in the cell was investigated. The results showed that pCAMBIA3300-GFP was expressed in the cell membrane, nucleus, and cytoplasm of young tobacco leaves, while pCAMBIA3300-... GmPPT Expression in chloroplasts indicates that this gene functions in chloroplasts. Figure 7 ).

[0024] 2) GmPPT Analysis of expression patterns in different tissue sites Fresh soybean root, stem, leaf, flower, pod, and seed samples were collected for RNA extraction. Reverse transcription of RNA was performed using a ReverTra AceqPCR RT Master Mix. Real-time quantitative PCR was then performed using a SYBR Select Master Mix RT-PCR instrument to obtain the RNA from soybean samples. GmActin4 As an internal reference gene (primer 2), the results showed that... GmPPT The gene has a high expression level during seed development. Figure 8 ).

[0025] Example 4. Transformation of Agrobacterium rhizogenes with recombinant vector Transformation method of Agrobacterium rhizogenes K599: Remove K599 competent cells from the -80℃ ultra-low temperature freezer and place them on ice. After keeping them in an ice-water mixture, add 1 μg of the pre-constructed pCAMBIA3300- GmPPT The recombinant plasmid was rapidly and vigorously mixed thoroughly, then incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Subsequently, 700 μL of liquid LB was added, and the mixture was incubated at 28°C and 200 rpm for 2 h. After centrifugation at 5000 rpm for 1 min, 700 μL of the supernatant was discarded. The remaining liquid was mixed by pipetting, and 100 μL was evenly spread onto solid LB medium (containing Kan and Str antibiotics). The medium was then incubated upside down at 28°C for 24 h. Single colonies were then picked for further colony PCR verification (primer 1). The target band of 1191 bp was obtained, confirming that pCAMBIA3300- GmPPT Successfully transferred into Agrobacterium rhizogenes K599 ( Figure 9 ).

[0026] Example 5. Obtaining Agrobacterium rhizogenes-mediated transgenic lines Select plump, smooth, and disease-free Dongnong 50 soybean seeds and place them in a desiccating bottle. Add 96 mL of NaClO and 6 mL of concentrated HCl, quickly seal the bottle, and place it in a fume hood. After sterilization for 16 hours, place the bottle in a pre-sterilized laminar flow hood to remove residual chlorine, and seal it with sealing film for preservation. Then, sow the sterile Dongnong 50 soybeans on germination medium for about one week. Next, spread 200 μL of Agrobacterium on YEP solid medium (containing Kan and Str antibiotics) and incubate in an inverted 28°C incubator for 24 hours. Resuspend the Agrobacterium in liquid CCM medium to OD. 600 =0.8, this liquid was used as the infection solution. Seedlings cultured for one week were used, and the seed coat and apical bud were removed in a sterile laminar flow hood. The explants were then infected using the infection solution. After infection, excess infection solution was blotted dry with filter paper, and the explants were placed on a co-culture solid medium and cultured in the dark for approximately 3 days. After dark culture, the explants were washed three times with sterile water containing Cef, thoroughly dried, and then placed in a rooting medium. After approximately 2 weeks of culture, hairy roots emerged, and positive identification was performed using Bar test strips. RNA was extracted from transgenic and control hairy roots to determine the expression level of the GmPPT gene in the overexpressing hairy roots. The results showed that the expression level of the GmPPT gene in the hairy roots of the overexpression lines (OE1 and OE2) was significantly higher than that in the control lines. The expression level of the GmPPT gene in the overexpression line OE1 was approximately 9 times higher than that in the control line, and the expression level of the GmPPT gene in OE2 was approximately 12 times higher than that in the control line, indicating that the construction of the overexpression lines was successful. The roots of the identified transgenic positive lines were further dried and preserved for subsequent fatty acid content determination.

[0027] Example 6. Functional identification of candidate genes Fatty acid content was determined using gas chromatography. First, dried transgenic hairy roots were ground into powder, and 0.1 g was weighed for later use. 3 mL of n-hexane was added, and the mixture was extracted in a 50℃ water bath for 30 min. After extraction, 3 mL of 0.5 mol / L sodium hydroxide methanol solution was added, and the mixture was shaken for 20 min. The mixture was allowed to stand at room temperature for 1 h, and the supernatant was filtered into a brown sample vial. Fatty acid content was determined using an Agilent gas chromatograph. The program settings were: initial temperature 180℃, hold for 1.5 min; temperature increased to 225℃ at 10℃ / min, hold for 2 min; injection port temperature 250℃; column flow rate 67.5 mL / min; split ratio 20:1; injection volume 1 μL. Five plants from each line were selected as biological replicates to measure the fatty acid content of transgenic soybean hairy roots. Data analysis was performed using GraphPad Prism software. The results showed that the total fatty acid content in the hairy roots of the control line was 0.691 mg / mL, while the total fatty acid content in the hairy roots of the overexpression lines OE1 was 0.763 mg / mL and OE2 was 0.761 mg / mL. Furthermore, the total fatty acid content of the overexpression lines (OE1 and OE2) was significantly higher than that of the control line. Figure 10 ).

Claims

1. Use of the amino acid sequence represented by SEQ ID NO. 8 in improving the total fatty acid content of soybean.

2. Use of overexpression of the gene represented by SEQ ID NO. 7 in improving the total fatty acid content of soybean.

3. Use of a recombinant vector containing the gene represented by SEQ ID NO. 7 in improving the total fatty acid content of soybean.

4. Use according to claim 3, characterized in that, The starting vector of the recombinant vector is pCAMBIA3300.

5. Use of a recombinant microorganism containing the gene represented by SEQ ID NO. 7 in improving the total fatty acid content of soybean.

6. Use according to claim 5, characterized in that, The starting microorganism of the recombinant microorganism is a prokaryotic microbial cell.

7. A breeding method for increasing the total fatty acid content of soybean, characterized by, The specific steps of the breeding method are as follows: Step 1: The gene represented by SEQ ID NO. 7 is connected with an overexpression vector to obtain a recombinant vector; Step 2: The recombinant vector of step 1 is transformed into Agrobacterium to obtain a recombinant Agrobacterium; Step 3: The recombinant Agrobacterium of step 2 is introduced into sugar beet to obtain transgenic soybean hairy roots, and after identification, positive transgenic soybean hairy roots are obtained.

8. The breeding method according to claim 7, characterized in that, The primer pair for amplifying the gene represented by SEQ ID NO. 7 in step 1 is SEQ ID NO. 1 and SEQ ID NO.

2.

9. The breeding method according to claim 7, characterized in that, The overexpression vector is pCAMBIA3300.

10. A method of increasing the total fatty acid content of soybean, characterized by, The gene represented by SEQ ID NO. 7 is overexpressed in soybean to obtain transgenic soybean.

Images