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CN-122012597-A - Application of soybean GmSUC gene in improving soybean seed protein content

CN122012597ACN 122012597 ACN122012597 ACN 122012597ACN-122012597-A

Abstract

The invention discloses an application of soybean GmSUC gene in improving soybean seed protein content, wherein the nucleotide sequence of soybean Gm SUC2 gene is shown as SEQ ID NO. 1, and the amino acid sequence of soybean GmSUC protein is shown as SEQ ID NO. 2. The invention creates GmSUC2 functional deletion mutant strain by using a gene editing method, and discovers that the protein content of the gmsuc mutant is obviously improved compared with the wild type. Therefore, the GmSUC gene knockout in soybean is one of effective ways for improving the protein content of soybean seeds, and has important production and theoretical significance for the breeding of soybean dominant varieties and the development of germplasm resources.

Inventors

  • XU DONGQING
  • SONG ZHAOQING
  • HU QING

Assignees

  • 南京农业大学

Dates

Publication Date
20260512
Application Date
20260316

Claims (10)

  1. 1. Use of a gene encoding soybean GmSUC protein to inhibit or knock out soybean 3832 protein in soybean breeding to increase soybean seed protein content or to cultivate high protein content, characterized in that the amino acid sequence of soybean GmSUC protein is (A1) or (A2) or (A3) as follows: (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-terminus and/or C-terminus of (A1) or (A2); preferably, the nucleotide sequence of the gene encoding soybean GmSUC protein is shown as SEQ ID NO. 1.
  2. 2. Use of a gene encoding soybean GmSUC protein to inhibit or knock out in the preparation of a product for increasing the protein content of soybean seeds or in the preparation of a product for breeding soybeans with high protein content, said soybean GmSUC protein having the amino acid sequence of (A1) or (A2) or (A3) as follows: (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-terminus and/or C-terminus of (A1) or (A2); preferably, the nucleotide sequence of the gene encoding soybean GmSUC protein is shown as SEQ ID NO. 1.
  3. 3. The use according to claim 1 or 2, wherein the suppression or knockout of the gene encoding soybean GmSUC protein is achieved by editing the GmSUC gene using the CRISPR/Cas9 system.
  4. 4. Use of a substance that suppresses or knocks out a gene encoding soybean GmSUC protein in soybean seed protein content improvement or soybean breeding for high protein content cultivation, characterized in that the amino acid sequence of soybean GmSUC protein is (A1) or (A2) or (A3) as follows: (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-terminus and/or C-terminus of (A1) or (A2).
  5. 5. Use of a substance that inhibits or knocks out a gene encoding soybean GmSUC protein in the preparation of a product that increases the protein content of soybean seeds or in the preparation of a product that breeds soybean with a high protein content, said soybean GmSUC protein having the amino acid sequence of (A1) or (A2) or (A3) as follows: (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-terminus and/or C-terminus of (A1) or (A2).
  6. 6. The use according to claim 4 or 5, wherein the substance inhibiting or knocking out the gene encoding soybean GmSUC protein is selected from siRNA against the gene encoding soybean GmSUC protein, gene editing system. Preferably, the target point of the gene editing system is SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6. More preferably, the gene editing system is obtained by inserting a double strand obtained by annealing SEQ ID NO.7 and SEQ ID NO.8, a double strand obtained by annealing SEQ ID NO.9 and SEQ ID NO.10, a double strand obtained by annealing SEQ ID NO.11 and SEQ ID NO.12 or a double strand obtained by annealing SEQ ID NO.13 and SEQ ID NO.14 into a pCas (EF 1A2 promoter) vector.
  7. 7. A method for increasing the protein content of soybean seeds, comprising decreasing the amount and/or activity of GmSUC protein as defined in claim 1 or decreasing the expression level of GmSUC gene.
  8. 8. A method of breeding soybeans with high protein content, comprising reducing the content and/or activity of GmSUC protein or reducing the expression level of GmSUC gene.
  9. 9. The method according to claim 7 or 8, wherein the reduction of the expression level of GmSUC gene is achieved by transferring a gene editing vector for GmSUC protein encoding gene into soybean, which is specifically constructed by editing GmSUC gene using CRISPR/Cas9 system, and ligating primer for GmSUC gene target to pCas (EF 1A2 promoter) vector to obtain recombinant plant expression vector; Preferably, the primer is selected from any one of the following: (I) SEQ ID NO.7 and SEQ ID NO.8; (II) SEQ ID NO.9 and SEQ ID NO.10; (III) SEQ ID NO.11 and SEQ ID NO.12; (IV) SEQ ID NO.13 and SEQ ID NO.14.
  10. 10. The primer set forth in claim 9.

Description

Application of soybean GmSUC gene in improving soybean seed protein content Technical Field The invention belongs to the field of biotechnology, and in particular relates to application of GmSUC gene in improving soybean seed protein content Background Soybean is a globally important vegetable protein source, the water-soluble protein content of the seeds directly determines the nutrition quality and processing performance of the soybean product, and synchronous improvement of the soybean protein content and yield is one of the core targets in the breeding field. However, regulating soy protein content is affected by polygenic control and environmental factors, and thus increasing soy protein content is a significant challenge. In addition, soybean is used as an ancient tetraploid, and multiple copies of genes exist, so that the genetic redundancy is limited. The transgenic technology plays an important role in solving the shape coordination improvement aspects of yield, quality and the like which are difficult to overcome by the conventional breeding technology. Sucrose is the major form of transport of most plant carbohydrates from source tissues to sink tissues and plays an important role in plant growth. Sucrose transporters (Sucrose transporters, SUT/cross carriers, SUC), which have a transmembrane domain of typically 12 a-helical structures, are responsible for the loading of Sucrose into the phloem and mediate its long distance transport. After the arabidopsis AtSUC2 is knocked out, sucrose in leaves of a mutant plant cannot be normally transported to other parts, so that a large amount of starch and soluble sugar are accumulated in the leaves, and the growth and development of the plant are blocked. After the spinach SoSUT1 is overexpressed in the potato, the output of sucrose in the leaf is promoted, and the content of soluble sugar in the tuber is obviously increased. In corn, inhibition of ZmSUT1 expression causes accumulation of soluble sugars in leaves in large amounts, leading to premature leaf senescence and delayed flowering. However, there is no disclosure in soybean that SUC is reported to be involved in the regulation of grain protein content. Therefore, the key members for regulating and controlling the protein content of the seeds in the soybean SUC family are mined, the functions of the key members are clarified and the key members are applied to molecular breeding, so that the key members have important significance in enriching the gene resources of soybean high-protein breeding and improving the breeding efficiency, and a new technical path can be provided for cultivating new varieties of high-quality high-yield soybeans. Disclosure of Invention Aiming at the defects of the prior art, the invention aims to provide an application of soybean GmSUC2 genes in improving the protein content of soybean seeds. In order to achieve the aim of the invention, the method can be realized by the following technical scheme: In a first aspect, the invention provides the use of a gene encoding soybean GmSUC protein for protecting and inhibiting or knocking out the soybean 3834 protein in soybean seed protein content improvement or soybean breeding for high protein content cultivation, wherein the amino acid sequence of soybean GmSUC protein is as follows (A1) or (A2) or (A3): (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-terminus and/or C-terminus of (A1) or (A2). Preferably, the nucleotide sequence of the gene encoding soybean GmSUC protein is shown as SEQ ID NO. 1. It will be appreciated by those skilled in the art that proteins obtained by performing one or more conservative amino acid substitutions (e.g., substitutions between amino acids of similar nature) on the sequence shown in SEQ ID NO.2 are intended to be within the scope of the present invention, as long as they are still knocked out and result in an increase in seed protein content. In a second aspect, the invention provides the use of a gene encoding soybean GmSUC protein for the preparation of a product for increasing the protein content of soybean seeds or for the preparation of a product for breeding soybeans for breeding high protein content, wherein the amino acid sequence of soybean GmSUC protein is as follows (A1) or (A2) or (A3): (A1) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2 in a sequence table; (A2) A protein which is derived from (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO. 2 in the sequence table; (A3) A fusion protein obtained by ligating a protein tag to the N-