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Monochamus alternatus heat shock protein MaltHSP20-11 and coding gene and application thereof

A technology of heat shock protein, Monochamus alternatus, applied in the field of heat shock protein, can solve the problems of less research on HSP20, and achieve the effect of high application value

Active Publication Date: 2020-08-25
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on heat shock proteins in insects mainly focuses on the HSP70 and HSP90 families, while there are few studies on HSP20, the first line of defense against heat stress.

Method used

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  • Monochamus alternatus heat shock protein MaltHSP20-11 and coding gene and application thereof
  • Monochamus alternatus heat shock protein MaltHSP20-11 and coding gene and application thereof
  • Monochamus alternatus heat shock protein MaltHSP20-11 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning the heat shock protein MaltHSP20-11 gene of Monochamus alternatus

[0025] 1. Obtain the heat shock protein MaltHSP20-11 gene sequence of Monochamus alternatus

[0026] The experimental materials were taken from the monochamus monochamus raised in the laboratory. After the molting, the 4th instar larvae continued to be fed for 3 days, and then they were subjected to high temperature stress at 40°C for 3 hours, then quickly frozen in liquid nitrogen and stored in a -80°C refrigerator as the heat treatment group. At the same time, the feeding conditions were 25 The same-day-old larvae at ℃ were quick-frozen in liquid nitrogen and stored in a -80℃ refrigerator as a control group. Every 3 larvae were mixed into one sample, and each sample had 3 biological replicates. After the samples were prepared, they were sent to Shanghai Meiji Biological Co., Ltd. for transcriptome sequencing.

[0027] The Illumina sequencing platform was used to complete the trans...

Embodiment 2

[0069] Example 2: Bioinformatics analysis of the heat shock protein MaltHSP20-11 gene of Monochamus alternatus

[0070] ExPasy software shows that the heat shock protein MaltHSP20-11 gene of Monochamus alternatus is encoded by 176 amino acids, and its amino acid sequence is shown in SEQ ID NO.2; the molecular weight of the protein is 19.97kDa; the isoelectric point is 6.21, which is an acidic protein; It is a hydrophilic protein (-3.311-1.211).

[0071] CELLO subcellular localization showed that the protein was located in the nucleus.

[0072] BLAST comparison showed that the heat shock protein MaltHSP20-11 gene of Monochamus alternatus contains a metazoan_ACD domain, which is a member of the heat shock protein 20 family.

[0073] Through the MEGA6.0 software, based on 1000 comparisons, the heat shock protein MaltHSP20-11 gene of Monochamus alternata was compared with those from Coleoptera, Lepidoptera, Hemiptera, Diptera, Hymenoptera and Orthoptera using the neighbor-joining...

Embodiment 3

[0074] Example 3: Analysis of the expression pattern of the heat shock protein MaltHSP20-11 gene of Monochamus alternatus under high temperature stress

[0075] The method for obtaining the template cDNA is the same as "3, the synthesis of cDNA" in Example 1, and the Rnase-Free H 2 O diluted to 0.1 μg / μL.

[0076] The fluorescent quantitative PCR reaction primers were designed by NCBI (Primer designing tool) as follows:

[0077] HSP20-11-qF: 5′-GCCTCTCCATCCATCTCGAA-3′

[0078] HSP20-11-qR: 5′-GTTACTGTTGATCCCGTGTCG-3′

[0079] Construct the fluorescent quantitative PCR reaction system: Hieff qPCR SYBR Green Master Mix (Yesen, China) 10 μL, RNase-Free H 2 O 7.2 μL, upstream and downstream primers (10 μmol / L) each 0.4 μL, cDNA 2 μL. Obtain the melting curve after the amplification, and judge whether each pair of primers is specific and whether there are primer-dimers according to the presence or absence of a single peak.

[0080] The fluorescent quantitative PCR reaction was...

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Abstract

The invention discloses monochamus alternatus heat shock protein MaltHSP20-11 and a coding gene and application thereof, and belongs to the field of heat shock proteins. The overall length of the monochamus alternatus heat shock protein HSP20 gene provided by the invention is 531bp, and 176 amino acids are coded. Through a luminescence quantitation method, the expression feature of the gene underthe heat stress condition is obtained, and high temperature can obviously induce the raising of the gene, so that results indicate that the protein can exert important effect of the protein for monochamus alternatus larvae to resist high temperature stress. Finally, a prokaryotic expression carrier is constructed, and through an expression in vitro method, the purified monochamus alternatus heat shock protein MaltHSP20-11 is obtained successfully. The monochamus alternatus heat shock protein MaltHSP20 after purification with a Ni+ resin column is subjected to molecular chaperone activity verification, results indicate that the protein can effectively protect thermal coagulation of malic dehydrogenase. Therefore, the application has high application value, and provides a molecule basis forresearch of heat resistance of the monochamus alternatus.

Description

technical field [0001] The invention belongs to the field of heat shock proteins, and more specifically relates to a heat shock protein MaltHSP20-11 of Monochamus alternatus and its coding gene and application. Background technique [0002] Monochamus alternatus belongs to the order Coleoptera and the family Cerambycidae. It is not only an important borer pest in pine forests in southern my country, but also the main vector of the world's quarantine pest pine wood nematode. The infection cycle of Monochamus alternata and B. xylophilus destroys the transport tissue of pine trees, leading to pine wilt disease, which poses a huge threat to forest resources in my country and the world. Monochamus alternatus is mainly distributed in the subtropical and tropical regions of my country. Due to global warming and extreme high temperatures, this pest sometimes experiences high temperatures as high as nearly 40°C in summer, and has strong heat resistance. [0003] In the long-term evo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11C12N15/70C12N1/21C12N9/04C12N9/96C12R1/19
CPCC07K14/43563C12N15/70C12N9/0006C12Y101/01037C12N9/96
Inventor 李慧陈文霞许高娟郝德君夏小洪
Owner NANJING FORESTRY UNIV
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