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CheY2 mutant protein and application thereof

A mutant and protein technology, which is applied in the application and use of vectors to introduce foreign genetic material, peptide sources, etc., can solve the problems of poor repeatability of related chemotaxis experiments and insufficient ability to adjust chemotaxis responses.

Active Publication Date: 2020-10-20
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the ability of natural Agrobacterium to regulate chemotaxis responses to chemotaxis attractants (such as AS, but not limited to AS) is not significant enough, and the reproducibility of related chemotaxis experiments is poor, which is relevant for the laboratory to verify bacterial chemotaxis. Great impact on research activities

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  • CheY2 mutant protein and application thereof
  • CheY2 mutant protein and application thereof
  • CheY2 mutant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Analysis of CheY family genes in A. tumefaciens

[0134] In the genome of A. tumefaciens, there are two homologous cheY genes, cheY1 (atu0516) and cheY2 (atu0520). They are located on a chemotaxis operon in A. tumefaciens. Sequence comparison with CheY of Escherichia coli revealed that the homology of CheY1 sequence and CheY of E. coli was 31.78%, and the homology of CheY2 sequence and CheY sequence of E. coli was 32.31%. A comparison of CheY in Agrobacterium tumefaciens, CheY in Escherichia coli and CheY2 in Rhizobium revealed that the two CheY2 proteins maintain a high degree of identity or similarity in the motor binding surface (α4-β5-α5 domain) . The motor-binding surface of Escherichia coli CheY is considered to be an important site for chemotactic signal transduction. This suggests that these same sites may also play an important role in chemotactic signal transduction in Agrobacterium. Several key residues on the α4-β5-α5 surface of CheY in E. coli...

Embodiment 2

[0135] Example 2 Obtaining of C58ΔcheY1 strain and C58ΔY1Y2 strain

[0136] 2.1 Construction of the recombinant suicide vector containing the upstream and downstream homology arms of the target gene

[0137] Download the Agrobacterium C58 cheY1 gene nucleotide sequence SEQ ID NO: 17 from the gene database, use primers SEQ ID NO: 18-19 to amplify the 550 bp upstream sequence of the cheY1 gene, and use primers SEQ ID NO: 20-21 to amplify the cheY1 gene downstream Sequence 550bp, connected to suicide vector pEX18km; download the nucleotide sequence of Agrobacterium C58 cheY2 gene SEQ ID NO: 12, use primers SEQ ID NO: 13-14 to amplify 550bp of the upstream sequence of cheY2 gene, use primers SEQ ID NO: 15-16 Amplify 550bp of the downstream sequence of the cheY2 gene, and connect to the suicide vector pEX18km; 2.2 Screening of target gene deletion mutants

[0138] A. Electrotransfer the suicide vector carrying about 550 bp upstream and downstream of the cheY1 gene into the Agrobac...

Embodiment 3

[0142] Example 3 Construction of Plasmid DNA for Complementary Target Gene and Obtaining of WTY2 Strain

[0143] 3.1. Cloning of the target gene

[0144] Using primers cheY2-PF (SEQ ID NO: 22) and cheY2-PR (SEQ ID NO: 23), the genome of Agrobacterium tumefaciens was used as a template to amplify the cheY2 gene sequence. The circular DNA pUCA19 plasmid was enzymatically cut into linear plasmids by double digestion. Using a homologous recombinase kit, connect the two into a recombinant plasmid. details as follows;

[0145] Add 6 μL of the amplified product cheY2, 2 μL of the linearized double-digested plasmid DNA pUCA19, and then add 2 μL of 5X Ligation-Free Cloning MasterMix (the kit name is Ligation-Free Cloning System), mix well and place in ice bath for 0.5 h. After the ice bath, add 10 μL of the reaction solution to 100 μL of competent cells (from Qingke Biotechnology Co., Ltd.), place on ice for 0.5 h, heat shock at 42 °C for 90 seconds, add 1 mL of LLB liquid medium an...

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Abstract

The invention discloses a CheY2 mutant protein and application thereof. Specifically, at least one of amino acids, located in the 93rd position, 96th position, 107th position, 109th position and 123rdposition of a wild chemotactic response regulatory protein, of the CheY2 mutant protein mutates. Compared with a wild CheY2 protein, the CheY2 mutant protein can remarkably improve swimming capacityof agrobacteria on a swimming flat plate, remarkably improve chemotactic capacity for a single substance acetosyringone (AS), but not limited to AS, in capillary tubes, and remarkably improve bindingcapacity of the CheY2 protein and FliM protein in the flagellar motor regulating area.

Description

technical field [0001] The invention belongs to the field of microorganism application, and relates to an Agrobacterium CheY2 mutant protein and its application. Background technique [0002] Agrobacterium tumefaciens is a Gram-negative bacterium that can transfer and integrate a DNA sequence (also known as T-DNA) on its own Ti plasmid into the genome of the host plant, resulting in the production of crown gall tumors and hereditary Transform the host. Therefore, people insert the required target gene into the modified T-DNA region, and use the infection of Agrobacterium tumefaciens to realize the transfer and integration of the exogenous gene to the plant cell, thereby obtaining the transgenic plant. At present, Agrobacterium tumefaciens has become an important tool for plant transgenics. Through the genetic transformation technology mediated by Agrobacterium tumefaciens, many important agricultural and horticultural crops have been successfully transformed, providing a g...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/74C12N1/21C12R1/01
CPCC07K14/195C12N15/743
Inventor 郭敏亮高大伟宗仁杰叶竞阳徐楠
Owner YANGZHOU UNIV
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