Soybean subtilisin gene and its application

A technology of Bacillus subtilis and protease, applied in the field of molecular biology, can solve problems that have not been reported

Active Publication Date: 2019-11-19
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 subtilisin gene and its application
  • Soybean subtilisin gene and its application
  • Soybean subtilisin gene and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning 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 And sequenced, the PCR amplification primers required for the test are shown 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...

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 soybean subtilisin gene Gmsbt1.6-3, which is the soybean subtilisin gene Gmsbt1.6-3 isolated and cloned from soybean root mutant material M18, and constructs a plant overexpression vector of the gene And RNAi expression vector, through the transformation of soybean plants, the transformed plants have the function of resisting Phytophthora root rot, and provide genetic resources and basic materials for the cultivation of new varieties resistant to 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 Patents(China)
IPC IPC(8): C12N9/50A01H5/00A01H6/54
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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