Recombinant vector and expression method of ustilago maydis effector protein CMU1 gene

A technology of corn smut and effector protein, applied in the biological field, can solve the problems of low success rate, cumbersome steps and the like

Pending Publication Date: 2019-11-08
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Recombinant vector and expression method of ustilago maydis effector protein CMU1 gene
  • Recombinant vector and expression method of ustilago maydis effector protein CMU1 gene
  • Recombinant vector and expression method of ustilago maydis effector protein CMU1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction and identification of the recombinant vector of the CMU1 gene of the effector protein of Corn smut

[0030] 1. Extract maize RNA and reverse transcribe cDNA

[0031] Take the corn smut material, use the RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the total RNA at the seedling stage, and use the reverse transcription kit (Promega) to reverse transcription to obtain cDNA.

[0032] 2. Amplify CMU1 gene with cDNA as template;

[0033] Design primers and use cDNA as a template to amplify the CMU1 gene. The nucleotide sequence of the CMU1 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the CMU1 gene is shown in SEQ ID NO. 2.

[0034] The primers are as follows:

[0035] Upstream primer: CMU1-F: GC GAATTC ATGGCGGCTGTATCTGGC underlined is EcoRI restriction site;

[0036] Downstream primer: CMU1-R: CG CTCGAG TTAGGTGCACTTGTTGGCG underlined is the XhoI restriction site;

[0037] The upstream an...

Embodiment 2

[0043] Example 2: Induced expression of CMU1 protein

[0044] 1. Obtain the recombinant prokaryotic expression strain of CMU1

[0045] The single clones successfully sequenced in Example 1 were inoculated into 50ug / mL kanamycin liquid medium and cultured overnight at 37°C and 200rpm. According to the plasmid extraction kit of Nanjing Novozan Biotechnology Co., Ltd., pET-28a -The CMU1 recombinant expression vector was extracted, and the recombinant expression vector was taken to transform E. coli BL21(DE3), Rosetta(DE3), BL21(DE3) pLysS strains, and the expression of CMU1 protein was detected.

[0046] 2. Cultivate activated strains overnight

[0047] The above recombinant prokaryotic expression strain was cultured overnight. For example, transfer the BL21(DE3) strain to 50ug / mL kanamycin liquid medium, and transfer Rosetta(DE3), BL21(DE3) pLysS strains to 50ug / mL kanamycin+50ug / mL chlorine The activated strain was cultured overnight at 37°C and 200 rpm in a liquid medium of mycin.

...

Embodiment 3

[0056] Example 3: Verification of CMU1 protein induced expression conditions

[0057] 1. Obtain the recombinant expression strain of CMU1

[0058] The single clones successfully sequenced in Example 1 were inoculated into 50ug / mL kanamycin liquid medium and cultured overnight at 37°C and 200rpm. According to the plasmid extraction kit of Nanjing Novozan Biotechnology Co., Ltd., pET-28a -CMU1 recombinant expression vector was extracted, and the recombinant expression vector was taken to transform E. coli BL21(DE3), Rosetta(DE3), BL21(DE3) pLysS strains, and spread on 50ug / mL kanamycin (Rosetta(DE3). Add 50ug / mL chloramphenicol) overnight culture at 37℃, pick a single colony and inoculate it into 50ug / mL kanamycin liquid medium, cultivate overnight at 37℃, 200rpm, add sterilized 50 % Glycerol, mixed and stored in a refrigerator at -80°C to obtain a prokaryotic expression strain expressing CMU1.

[0059] 2. Determination of the best strain for CMU1 gene induction and expression

[0060...

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Abstract

The invention discloses a recombinant vector for expressing an ustilago maydis effector protein CMU1 gene and further discloses a method for expressing the ustilago maydis effector protein CMU1 gene.A prokaryotic expression vector of the ustilago maydis effector protein CMU1 gene is constructed, a series of optimization is performed on protein expression conditions, CMU1 soluble protein is finally obtained, and the result lays a foundation for further study on crystal structures and biological characteristics of protein, provides a thought for designing and developing pesticides targeting CMU1 and further provides a basis for control of ustilago maydis.

Description

Technical field [0001] The invention relates to a recombinant vector and an expression method of the CMU1 gene of the effector protein of corn smut fungus, and belongs to the field of biotechnology. Background technique [0002] Corn smut is one of the main diseases of corn, which can occur throughout the growth period of corn. However, the corn smut can be cooked or eaten raw when it is young. It has a sweet taste and fried food has a different flavor. Regular consumption can prevent and treat liver system and gastrointestinal ulcers, and can help digestion and laxation. Corn smut contains 16 kinds of amino acids including glutamic acid, lysine, alanine, arginine, methionine, threonine and histidine. This corn smut can be processed for medicinal purposes. Fresh spore piles are picked off or old mature honey pills are collected. It is cold in nature and sweet in taste. It is beneficial to the liver and stomach, and it can also cure neurasthenia and children. Malnutrition. [00...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C07K14/37
CPCC12N15/70C12N15/66C07K14/37
Inventor 谢鑫李向阳蒋君梅王勇任明见孙涛
Owner GUIZHOU UNIV
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