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RNAi plant expression vector of soybean allergenic protein gene Gly m Bd 30 K

A plant expression vector and protein technology, applied in the direction of DNA / RNA fragments, plant products, recombinant DNA technology, etc., can solve the problems of limited utilization space, limited soybean genetic resources, time-consuming, etc., and achieve the effect of eliminating harm

Inactive Publication Date: 2010-12-22
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Although traditional breeding techniques have successfully improved the nutritional value of soybeans, this process is not only time-consuming, but also has limited utilization space, and it is difficult to change multiple quality traits of soybeans at the same time. After all, the genetic resources that can be improved in soybeans are limited. , and the improvement of soybean traits is limited to the mutant materials that have been obtained

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  • RNAi plant expression vector of soybean allergenic protein gene Gly m Bd 30 K
  • RNAi plant expression vector of soybean allergenic protein gene Gly m Bd 30 K
  • RNAi plant expression vector of soybean allergenic protein gene Gly m Bd 30 K

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Embodiment Construction

[0037] Build strategy:

[0038] 1> Selection of introns—select the intron sequence of the GUS gene in the commonly used expression vector PCAMBIA1301 as the middle fragment of the interference vector;

[0039] 2> Selection of Gly m Bd 30K interference fragments—Use BLAST database comparison to select fragments that are located in the Gly m Bd 30K coding region and have low homology to other genes in the soybean genome;

[0040] 3> Selection of the insertion site of the hairpin structure—introduce the hairpin structure behind the His-tag terminator TGA of the GUS gene of the PCAMBIA1301 vector.

[0041] 4> The pattern of the hairpin structure is: antisense fragment + intron + sense fragment

[0042] Construction process of Gly m Bd 30K RNAi vector pCAscil-1301:

[0043] 1. Cloning of Gly m Bd 30K interference fragment:

[0044] Select 'Nannong 32' (approved variety, approval number: National Approved Bean 2008025, purchased in the market) as material, take young tender seeds...

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Abstract

The invention relates to a construction method of an RNAi plant expression vector of a soybean allergenic protein gene Gly m Bd 30 K, belonging to the field of molecular biology. The method comprises the following steps: respectively designing a primer amplification Gly m Bd 30 K interference segment and an intron fragment to be connected with a pMD19-T vector, and transforming TOP10; and introducing restriction sites at two ends, connecting three fragments [30k-antisense(A), intron(I) and 30k-sense(S)] together, and connecting the A-I-S structure into pCAMBIA1301 to form an interference vector pCAScil-1301. The soybeans transformed by the RNAi plant expression vector carry out gene silencing on allergenic protein genes, thereby laying a solid foundation for ensuring that allergenic people eat soybeans safely. The invention has important theoretical and practical significance for improving the technical level of soybean molecular breeding in China.

Description

1. Technical field [0001] The invention relates to the construction of an RNAi plant expression vector of soybean allergy protein gene Gly m Bd 30K, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] RNA interference (RNAi), also known as post-transcriptional gene silencing, is a new way to quickly turn off genes. It is also a new technology to study the gene expression, regulation and function of organisms. Its essence is the specific silencing of homologous genes in biological cells caused by small interfering RNA (siRNA). Its mechanism of action is that siRNA specifically binds to the corresponding mRNA and causes degradation, thereby preventing the translation of mRNA and specifically inhibiting the expression of the target gene, thus becoming a powerful tool for gene knockout. At present, with the continuous deepening of research, the mechanism of RNAi is being gradually elucidated. At the same time, as a powerful tool in ...

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Application Information

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IPC IPC(8): C12N15/82C12N15/113A01H5/00
Inventor 朱月林盖钧镒刘思辰杨立飞
Owner NANJING AGRICULTURAL UNIVERSITY
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