Soybean type bacillus subtilis proteinase gene and application

A technology of Bacillus subtilis and protease, applied in the field of molecular biology, can solve problems such as unreported

Active Publication Date: 2017-05-10
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the functional research of the subtilisin-like protease gene in soybean and its relationship with Phytophthora root rot.

Method used

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  • Soybean type bacillus subtilis proteinase gene and application
  • Soybean type bacillus subtilis proteinase gene and application
  • Soybean type bacillus subtilis proteinase gene and application

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Experimental program
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Effect test

Embodiment 1

[0029] The clone of the cDNA sequence of embodiment 1 gene Gmsbt1.6-3

[0030] The soybean (Glycine max) variety Jinong 18 and the soybean root mutant material M18 were provided by the Plant Biotechnology Center of Jilin Agricultural University.

[0031] Cloning of cDNA sequence of gene Gmsbt1.6-3

[0032] Total RNA was extracted from the root tissue of soybean M18 seedlings using the RNAiso kit produced by TAKARA, and the reverse-transcribed cDNA was used as a template. According to the sequence of RNA-seq sequencing results, specific primers sbt were designed, and the target fragment was amplified by RT-PCR The PCR amplification primers required for the test are listed in Table 1.

[0033] Table 1 PCR primer sequences

[0034]

[0035] According to the Gmsbt1.6-3 sequence fragment obtained in the previous screening, a specific primer sbt with an amplification length of 764bp was designed, and after PCR amplification, a nucleic acid band with the same size as expected wa...

Embodiment 2

[0036] Example 2 Gmsbt1.6-3 Gene bioinformatics analysis and phylogenetic tree construction

[0037] Will Gmsbt1.6-3 The full sequence of the gene was connected into the cloning vector pMD18-T Vector, and the recombinant vector was sequenced for bioinformatics analysis. The sequencing was completed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

[0038] Use the online analysis program ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to analyze the possible open reading frame of the new gene sequence, and translate the new gene ORF into an amino acid sequence through the DNAMAN software; use ProtParam Software (http / / www. web. expasy. org / protparam / ) to predict theoretical parameters of protein sequences; constructed using SWISS-MODEL online software Gmsbt1.6-3 The three-dimensional protein structure of the gene; use Protscale software (http: / / web.expasy.org / / protscale / to predict the hydrophilicity and hydrophobicity of the protein.

[0039] Download all known s...

Embodiment 3

[0044] Construction and genetic transformation of embodiment 3 plant expression vector

[0045] restriction endonuclease Bgl II and Pml I carried out double digestion of the plant expression vector pCAMBIA3301, and utilized the Seamless Assembly Clonng Kit kit to clone the obtained Gmsbt1.6-3 The full-length gene replaces the original one on 3301 GUS Gene. build with bar As a screening marker, the CaMV35S promoter promotes the target gene Gmsbt1.6-3 pCAMBIA3301- Gmsbt1.6-3 Plant overexpression vector.

[0046] use Bgl II and Pml The plant expression vector pCAMBIA3301 was double-digested with I restriction endonuclease to obtain a large fragment of the 3301 vector of 10kb and a GUS gene fragment of 2029bp; the cloning vector Pmd18-T-Gmsbt1.6-3 was carried out by using primer sbt-over PCR amplification, the Gmsbt1.6-3 gene ORF full-length fragment with a size of 591bp was obtained ( Figure 4 ), using T4 ligase to connect the full-length ORF fragment of Gmsbt...

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Abstract

The invention provides a soybean type bacillus subtilis proteinase gene Gmsbtl.6-3. The soybean type bacillus subtilis proteinase gene Gmsbtl.6-3 is separated and cloned from a soybean root system mutant material M18, and a plant over-expression vector and an RNAi (RNA (Ribonucleic Acid) interfere) expression vector of the gene are constructed; soybean plants are converted and the converted plants have a phytophthora root rot resisting function; gene resources and basic materials are provided for culturing new varieties capable of resisting soybean phytophthora root rot.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to soybean subtilisin gene (Gmsbt1.6-3) and its application. Background technique [0002] Serine protease (Serine protease) is a kind of proteolytic enzyme whose functional domain takes serine as the active center, and acts as a regulator by activating or inhibiting the protease. Serine protease is an important proteolytic enzyme, there are about 30 families, belonging to six categories: chymotrypsin, subtilisin-like protease, carboxypeptidase C, alanine protease, repressor protein LexA, and adenosine triphosphate-dependent serine protease. Among them, the subtilisin-like protease family is one of the largest families of serine proteases. [0003] Subtilisin-like proteases are widely found in organisms such as bacteria, fungi and parasites. This type of protease has a typical Asp / Ser / His catalytic domain in structure and has a wide range of physiological functions. St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50A01H5/00
CPCC12N9/63C12N15/8282
Inventor 王丕武宋阳张学明王鑫雨姚丹刘思言张卓金羽琨
Owner JILIN AGRICULTURAL UNIV
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