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Expression vector for improving soy protein content and quality as well as preparation and use thereof

A technology of soybean protein and expression vector, which is applied in the field of plant genetic engineering, can solve the problems of allergic reactions and the inability to increase the expression of 11S globulin in a targeted manner, and achieve the effect of avoiding blindness

Inactive Publication Date: 2008-08-27
张敏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0027] The technical problem to be solved by the present invention is to provide an expression vector for improving soybean protein content and quality and its preparation method and use, so as to overcome the inability of the prior art to increase the expression of a certain high-quality 11S globulin and the use of different Source protein gene may lead to defects in human food allergic reactions

Method used

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  • Expression vector for improving soy protein content and quality as well as preparation and use thereof
  • Expression vector for improving soy protein content and quality as well as preparation and use thereof
  • Expression vector for improving soy protein content and quality as well as preparation and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0060] a) According to the glycinin Gy7 gene sequence published by Genbank AF319776, specific PCR primers were designed, one end of the upstream primer was introduced into the XbaI site, and one end of the downstream primer was introduced into the XhoI site; Utilize conventional PCR to amplify about 2.2kb part after the signal peptide of glycinin Gy7 gene sequence; or extract mid-maturity soybean (Glycine max) seed RNA, design specific primers according to the glycinin Gy7 gene mRNA sequence published by GenbankAF319777, and introduce upstream and downstream primers respectively For the XbaI site and the XhoI site, use reverse transcription and PCR amplification to obtain the sequence after the signal peptide of the cDNA fragment of about 1.6kb Gy7 gene; connect the PCR product to the T vector to obtain the intermediate vector T-3, and transform competent Escherichia coli; The intermediate carrier T-3 was detected by conventional methods such as molecular biology enzyme digesti...

Embodiment 2

[0063] a) According to the same method as in Example 1, specific PCR primers were designed according to the glycinin Gy7 gene sequence published by Genbank AF319776, an XbaI site was introduced at one end of the upstream primer, and an XhoI site was introduced at one end of the downstream primer. amplify about 2.25kb glycinin Gy7 gene sequence (starting from ATG, with Gy7 gene signal peptide) from the total DNA; or extract mid-maturity soybean (Glycine max) seed RNA, according to the soybean ball published by Genbank AF319777 Specific primers were designed for the mRNA sequence of the protein Gy7 gene, and XbaI sites and XhoI sites were introduced into the upstream and downstream primers respectively, and about 1.65 kb of the Gy7 gene cDNA sequence was obtained by reverse transcription and PCR amplification (also starting from ATG, with the Gy7 gene signal peptide); the PCR product was connected with the T carrier to obtain the intermediate carrier T-4, and transformed into com...

Embodiment 3

[0066] In the pCAMBIA1300 plasmid vector (see figure 1 As shown) PCR primers were designed downstream of the 35S gene terminator, and a PstI restriction site was introduced at one end; using this primer and the primer (also with a PstI restriction site) previously paired with the upstream sequence of the Gy1 gene promoter (also with a PstI restriction site) for the expression in Example 1 The constructed target vector-p1300-Gy1-S1-Gy7(1) vector was amplified by PCR to obtain the Gy1 gene promoter, signal peptide, Gy7 gene sequence and 35S gene terminator, and the PCR product was connected to the T vector to obtain vector 2 , and transformed into competent Escherichia coli; conventional methods such as molecular biology enzyme digestion and PCR were used to detect carrier 2; vector 2 was digested with PstI, and the large fragment after enzyme digestion was recovered, and combined with pCAMBIA1301 (referred to as p1301, from Shanghai, Chinese Academy of Sciences) Researcher Wang...

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Abstract

The invention discloses an expression vector which increases the content and the quality of soybean protein, a method for preparing the expression vector, and the application. The expression vector comprises a glycinin gene promoter, a glycinin gene fragment, a signal coding sequence, a terminator and an endonuclease sensitive sequence, wherein the glycinin gene promoter is a globulin gene promoter of a first group of soybeans 11S, and the glycinin gene fragment is a soybean 11S globulin Gy7 gene fragment. The expression vector of the invention can be applied to transform the soybeans to obtain high quality soybean 11S globulin new varieties with high content and the high quality soybean 11S globulin new varieties with high content and without resistance selective marker genes and without reporter genes. The expression vector of the invention avoids the blindness on breeding and the defect that the adoption of heterogeneous source protein genes leads to allergic reaction possibly when human bodies eat, and the content of polypeptide which is synthesized by high quality soybean 11S globulin Gy7 which is obtained through transforming is at least increased by one time than control soybean which is not transformed.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an expression carrier for improving soybean protein content and quality, its preparation method and application. Background technique [0002] Soybean is one of the important crops in my country. It is rich in protein and 8 kinds of essential amino acids that cannot be synthesized by humans and animals. It is the main source of plant protein for human food and animal breeding. In recent years, the Food and Agriculture Organization of the United Nations has strongly advocated the development of soybean food to solve the current shortage of protein resources in developing countries. Therefore, improving soybean protein content and quality is one of the most important contents. [0003] At present, with the improvement of people's health and health awareness, the demand for high-quality high-protein soybean flour in the market is increasing year by yea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H1/00
Inventor 李建粤王帆逯慧顾婷玉肖刚石少华
Owner 张敏
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