Application of glycosyl phosphatidylinositol anchored lipid transporter GmLTPG in improvement of soybean grain traits

Abstract

The invention relates to the technical field of genetic engineering, and discloses application of glycosyl phosphatidylinositol anchored lipid transporter GmLTPG in improvement of soybean grain traits. The invention discloses an application of glycosyl phosphatidylinositol anchored lipid transporter protein GmLTPG in the aspect of improving soybean grain traits. Specifically, the invention provides an innovative method for improving the characters of the soybean seeds, especially for increasing the content of water-soluble protein in the soybean seeds and improving the oil quality. By means of the genetic engineering technology, the content of specific protein and grease in soybean seeds can be controlled more accurately and efficiently, so that the breeding period is shortened, and the breeding efficiency is improved. The implementation of the invention provides a new genetic resource for the field of soybean breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of a glycosylphosphatidylinositol-anchored lipid transport protein GmLTPG in improving soybean seed traits. Background Art

[0002] Soybean (Glycine max) is a key oil and protein crop. Studies have shown that soybean seeds are rich in high-quality protein and oil. Its protein contains a variety of essential amino acids for the human body, which has a significant positive impact on health. The high oil content of soybeans makes it an important source of vegetable oil, especially the rich unsaturated fatty acids such as linoleic acid, which are extremely beneficial to human health.

[0003] The characteristics of soybean seeds directly affect the yield and quality of soybeans. Traditionally, soybean breeding relies on hybridization and selection, but this method is time-consuming and inefficient. Advances in molecular biology technology have made it possible to improve crop characteristics through genetic engineering. The extraction efficiency of protein in soybean seeds mainly depends on the solubility of protein, that is, the content of water-soluble protein. This characteristic has become a key indicator for evaluating the utilization value of soybean seed protein. Using genetic engineering technology, molecular breeding can be carried out in soybeans to improve the quality characteristics of soybean seeds. The content of total protein, water-soluble protein and oil can be adjusted according to actual production needs to meet the needs of different production and processing companies. Summary of the invention

[0004] In view of this, the present invention proposes the application of glycosylphosphatidylinositol-anchored lipid transfer protein GmLTPG in improving soybean seed traits, aiming to provide a new way to improve the traits of soybean seeds, especially to increase the content of water-soluble protein and the quality of oil in soybean seeds.

[0005] The invention provides an application of a substance for improving the expression of a protein coding gene in improving the soybean protein content.

[0006] The protein is any of the following proteins:

[0007] B1) The amino acid sequence is the protein shown in SEQ ID NO:9;

[0008] B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1);

[0009] B3) connecting the N-terminus or / and C-terminus of B1) or B2) to a protein tag to obtain a fusion protein;

[0010] Preferably, the soybean is Williams82;

[0011] Preferably, the protein content is total protein;

[0012] More preferably, the protein content is water-soluble protein.

[0013] Further, the protein is derived from soybeans;

[0014] Preferably, the soybean is Williams82.

[0015] Furthermore, the substance that increases the expression of the gene encoding the protein is a nucleic acid molecule encoding the protein;

[0016] Preferably, the nucleic acid molecule sequence is sequence 10.

[0017] Furthermore, the substance that increases the expression of the gene encoding the protein is an expression cassette containing the above-mentioned nucleic acid molecule.

[0018] Furthermore, the substance for increasing the expression of the gene encoding the protein is a recombinant vector containing the above nucleic acid molecule or the above expression cassette.

[0019] Furthermore, the substance that increases the expression of the gene encoding the protein is a recombinant microorganism containing the above nucleic acid molecule, the above expression cassette, and the above recombinant vector.

[0020] Furthermore, the substance that increases the expression of the gene encoding the protein is a transgenic plant cell line containing the above nucleic acid molecule, the above expression cassette, and the above recombinant vector.

[0021] Furthermore, the substance that increases the expression of the gene encoding the protein is a transgenic plant tissue or organ containing the above nucleic acid molecule, the above expression cassette, and the above recombinant vector.

[0022] The present invention also provides the above protein.

[0023] The present invention also provides the above-mentioned substance for increasing the expression of the gene encoding the protein.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] It provides a new way to improve soybean seed traits, especially to increase the content of water-soluble protein and the quality of oil in soybean seeds, thereby increasing the nutritional value and commercial value of soybeans; through genetic engineering, the content of specific protein and oil in soybean seeds can be more accurately and efficiently regulated, shortening the breeding cycle and improving breeding efficiency.

[0026] The implementation of the present invention can provide new genetic materials for soybean breeding, which is helpful to cultivate soybean varieties that adapt to different environments and market demands; by increasing the content of water-soluble protein, the extraction efficiency and utilization value of soybean protein can be improved, which is of great significance to the food industry and feed industry; by improving the quality of oil, the unsaturated fatty acid content of soybean oil can be increased, thereby improving its competitiveness in the health food market;

[0027] The technical solution of the present invention has good application prospects and can be widely used in the genetic improvement of soybeans and other crops, providing new ideas and methods for crop quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0029] Figure 1 The verification results of positive transgenic soybean plants;

[0030] Figure 2 It is a statistical bar graph of the expression of GmLTPG gene in five strains of control Williams82 and GmLTPG gene overexpression plants;

[0031] Figure 3 The mature plant pictures of Williams82 control and five GmLTPG gene overexpression lines;

[0032] Figure 4 It is a statistical bar graph of the total protein content in grains of the five strains with overexpression of the Williams82 and GmLTPG genes;

[0033] Figure 5 It is a statistical bar graph of the water-soluble protein content in the grains of the five strains with overexpression of the Williams82 and GmLTPG genes;

[0034] Figure 6 It is a statistical bar chart of the oil content in grains of the five strains with overexpression of the Williams82 and GmLTPG genes. DETAILED DESCRIPTION

[0035] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to be able to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] The present invention discloses the application of the GmLTPG gene in regulating soybean seed traits. The amino acid sequence encoded by the GmLTPG gene is shown in SEQ ID NO.9. The specific sequence is as follows:

[0037] MGSKGAKNLALSSALLLLLVGFATSDINQDKAECTDKLLGLAGCLSYVGGEAKVPTMDCCSGIKEVINKSKRCLCILIKDRDDPSLGLKINVTLALNLPDVCETPTNITQCVDLLHLAPKSQEAKVFEGFEKALTNKTSPSPVLSANNTTAKGTSTSANNNSGGGWGKRWLVAEVVCVILPIVFISHFFLFLV*.

[0038] In the present invention, soybean seed quality traits include total protein, water-soluble protein and oil content in the seeds. The preferred method for regulating soybean seed traits includes increasing the expression of the GmLTPG gene to improve one or both of the total protein and water-soluble protein content of soybeans. The reagent for increasing the expression of the GmLTPG gene is preferably a recombinant vector containing the GmLTPG gene. The recombinant vector preferably includes the GmLTPG gene and the original vector, wherein the original vector is preferably a pCAMBIA3300 vector.

[0039] The nucleotide sequence of the GmLTPG gene is preferably as shown in SEQ ID NO.10, and the specific sequence is as follows: 5'--3'.

[0040] The soybean GmLTPG gene was cloned by PCR, and plants overexpressing GmLTPG were obtained by transgenic technology. The results showed that the total protein and water-soluble protein contents of the overexpressed transgenic soybeans were increased, while the oil content was reduced. The soybean glycosylphosphatidylinositol-anchored lipid transport protein gene GmLTPG provided by the present invention can be introduced into plants as a target gene to regulate the seed quality traits of transgenic plants, which is of great significance for the cultivation of new high-quality soybean varieties.

[0041] The present invention also discovered the important role of GmLTPG protein in affecting soybean seed quality traits, which is of great significance for clarifying the biological functions and molecular mechanisms of action of the glycosylphosphatidylinositol-anchored lipid transport protein family.

[0042] In addition, the present invention also provides the use of GmLTPG protein or GmLTPG gene or recombinant vector containing GmLTPG gene in soybean breeding. By introducing GmLTPG gene into soybean plants, new soybean varieties with increased total protein and water-soluble protein content and reduced oil content can be cultivated.

[0043] The present invention also provides a method for breeding high-quality soybean varieties, comprising overexpressing the GmLTPG gene in the soybean plant to be improved. In order to further illustrate the present invention, the application of the GmLTPG gene provided by the present invention in regulating soybean seed traits is described in detail below in conjunction with the accompanying drawings and examples, but these descriptions should not be regarded as limiting the scope of protection of the present invention.

[0044] Example 1 Construction of GmLTPG gene overexpression plants

[0045] RNA was extracted from soybean material Williams82 and reverse transcribed into cDNA. Then, PCR amplification was performed using primers F1 and R1 to obtain the amplified product. The primer sequences are as follows:

[0046] F1: 5'-tatgaccatgattacgaattcATGGGGTTCCAAAGGGGCAAAA-3', SEQ ID NO.1;

[0047] R1: 5'-aggtcgactctagaggatccTCAAACTAGGAACAAGAAAAAA-3', SEQ ID NO.2;

[0048] Among them, the lowercase sequence 5'-tatgaccatgattacgaattc-3' (SEQ ID NO. 11) and the lowercase sequence 5'-aggtcgactctagaggatcc-3' (SEQ ID NO. 12) were used as homology arms.

[0049] The amplification reaction system includes: cDNA 30-100 ng, F1 (10 μM) 2.5 μL, R1 (10 μM) 2.5 μL, PCR MasterMix 25 μL, and ddH2O is used to supplement the total volume to 50 μL.

[0050] The reaction procedure was as follows: the first step was pre-denaturation at 94°C for 5 minutes; the second step was denaturation at 94°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 1 minute, and this step was repeated for 30 cycles; the third step was final extension at 72°C for 5 minutes.

[0051] The expression vector pCAMBIA3300 was double-digested with EcoRⅠ and BamHⅠ to obtain a linearized vector; the expression vector pCAMBIA3300-GmLTPG was obtained by homologous recombination.

[0052] The expression vector pCAMBIA3300-GmLTPG was transformed into the soybean material Williams82 through the EHA105 Agrobacterium tumefaciens-mediated genetic transformation system to construct GmLTPG overexpression plants. The pCAMBIA3300 vector contains the selection marker gene EPSPS, which encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which can block the interference of glyphosate on the biosynthetic pathway, thereby protecting the plant from glyphosate killing.

[0053] The method for constructing and detecting overexpression plants is as follows:

[0054] (1) The obtained overexpression plants were transplanted into small pots and placed in a light incubator for cultivation. Fertilization and watering were carried out normally. When the plants grew to about 10 cm in height, they were transplanted to the field. After the plants grew up, leaves were taken for transgenic positive detection. The detection primers are as follows:

[0055] F2: 5'-GACGCACAATCCCACTATCC-3', SEQ ID NO.3;

[0056] R2: 5'-TTAATAATCTGAACTGAATGGT-3', SEQ ID NO.4;

[0057] If the amplified fragment size is 1656 bp, it indicates that the plant to be tested is a positive plant. The positive plants are harvested separately, and the homozygous transgenic plants are identified in the T2 generation, which are the plants overexpressing the GmLTPG gene. The electrophoresis verification results of the transgenic lines are shown in Figure 1 , where M is a 2000 bp marker, + is a plasmid positive control, - is a blank negative control, and 1-5 represent independent transgenic line samples OE-GmLTPG-1 to OE-GmLTPG-5, respectively.

[0058] (2) The relative expression of the GmLTPG gene in the control (OE-CK) and the five strains of the GmLTPG gene overexpressing plants was detected by fluorescence quantitative PCR. The fluorescence quantitative primer sequence of the GmLTPG gene is:

[0059] F3: 5'-ACAAAACTAGTCCCTCCCCG-3', SEQ ID NO.5;

[0060] R3: 5'-AATCACACACACCACCTCTGC-3', SEQ ID NO.6;

[0061] The internal reference gene was the soybean tubulin gene (Glyma.20G136000) (https: / / www.soybase.org / ), and the primer sequences were:

[0062] F4: 5'-CCTCGTTCGAATTCGCTTTTTG-3', SEQ ID NO.7;

[0063] R4: 5'-CAACTGTCTTGTCACTTGGCAT-3', SEQ ID NO. 8.

[0064] Results Figure 2 , data were analyzed by SPSS software for variance analysis (ANOVA), *** represents p < 0.001, ** represents p < 0.01, and * represents p < 0.05. Fluorescence quantitative PCR results showed that the expression level of GmLTPG in the positive overexpression strains was higher than that in the control material, about 1.24-6.63 times higher.

[0065] The total protein, water-soluble protein and oil content of the five strains of the control Williams82 (WT) and the GmLTPG gene overexpression plants were detected. The detection method of the above three traits was a near-infrared analyzer, the instrument name and model was Perton DA7250, and the results are shown in Figure 4-Figure 6 , where *** represents p < 0.001, ** represents p < 0.01, and * represents p < 0.05. The results showed that the total protein and water-soluble protein contents of the positive overexpression strains were higher than those of the control, with the total protein content increasing by 0.69-3.64%, the water-soluble protein content increasing by 2.28-4.26%, and the oil content decreasing by 0.61-1.35%.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. Use of a substance for increasing the expression of a protein encoding gene in increasing the protein content of soybeans, characterized in that: The protein is any of the following proteins: B1) The amino acid sequence is the protein shown in SEQ ID NO:9; B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1); B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that: The protein is derived from soy beans.

3. The use according to claim 1 or 2, characterized in that: The substance that increases the expression of the gene encoding the protein is a nucleic acid molecule encoding the protein according to claim 1 or 2.

4. The use according to claim 1 or 2, characterized in that: The substance for increasing the expression of the gene encoding the protein is an expression cassette containing the nucleic acid molecule according to claim 3.

5. The use according to claim 1 or 2, characterized in that: The substance for increasing the expression of the gene encoding the protein is a recombinant vector containing the nucleic acid molecule of claim 3 or the expression cassette of claim 4.

6. The use according to claim 1 or 2, characterized in that: The substance for increasing the expression of the gene encoding the protein is a recombinant microorganism containing the nucleic acid molecule of claim 3, the expression cassette of claim 4, and the recombinant vector of claim 4.

7. The use according to claim 1 or 2, characterized in that: The substance for increasing the expression of the gene encoding the protein is a transgenic plant cell line containing the nucleic acid molecule of claim 3, the expression cassette of claim 4, and the recombinant vector of claim 4.

8. The use according to claim 1 or 2, characterized in that: The substance for increasing the expression of the gene encoding the protein is a transgenic plant tissue or organ containing the nucleic acid molecule described in claim 3, the expression cassette described in claim 4, or the recombinant vector described in claim 4.

9. A protein according to claim 1.

10. A substance for increasing the expression of a gene encoding the protein according to any one of claims 3 to 8.

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