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A heat shock protein malthsp20-11 of Monochamus alternatus and its coding gene and application

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: 2022-06-28
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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  • A heat shock protein malthsp20-11 of Monochamus alternatus and its coding gene and application
  • A heat shock protein malthsp20-11 of Monochamus alternatus and its coding gene and application
  • A heat shock protein malthsp20-11 of Monochamus alternatus and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of the heat shock protein MaltHSP20-11 gene of P. pineapple

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

[0026] The experimental materials were taken from the laboratory-raised S. chinensis. After molting, the 4th instar larvae were continuously reared for 3 days, then subjected to high temperature stress at 40 °C for 3 h and then quickly frozen in liquid nitrogen and stored in a -80 °C refrigerator as a heat treatment group. At the same time, the feeding conditions were 25 The larvae of the same age at ℃ were quick-frozen in liquid nitrogen and stored in a -80 ℃ refrigerator as a control group. Each 3 larvae were mixed into a 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 transcriptome sequencing. After removing seque...

Embodiment 2

[0069] Example 2: Bioinformatics analysis of the heat shock protein MaltHSP20-11 gene of A. pineapple

[0070] ExPasy software showed that the heat shock protein MaltHSP20-11 gene of A. pineapple is encoded by 176 amino acids, and its amino acid sequence is shown in SEQ ID NO. For hydrophilic protein (-3.311-1.211).

[0071] The subcellular localization of CELLO shows that the protein is located in the nucleus.

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

[0073] By MEGA6.0 software, based on 1000 alignments, the heat shock protein MaltHSP20-11 gene of P. The phylogenetic tree constructed from the heat shock protein gene sequences of the order insects showed that the heat shock protein MaltHSP20-11 gene of P. .1) and HSP20-11 of Ceratocystis chinensis (QEL52520.1).

Embodiment 3

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

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

[0076] 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] Construction of real-time quantitative PCR reaction system: HieffqPCR 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. After the amplification, a melting curve was obtained, and whether each pair of primers was specific and whether there was a primer-dimer was judged according to the presence or absence of a single peak.

[0080] Real-time PCR reactions were performed on a 7500 Real-Time ...

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Abstract

The invention discloses a heat shock protein MaltHSP20‑11 of Monochamus alternatus and its coding gene and application, belonging to the field of heat shock proteins. The full length of the heat shock protein HSP20 gene of Monochamus alternatus provided by the invention is 531 bp, encoding 176 amino acids. The expression characteristics of this gene under heat stress conditions were obtained by fluorescence quantitative method, and high temperature can obviously induce the up-regulation of this gene, indicating that this protein plays an important role in the resistance of Monochamus alternata larvae to high temperature stress. Finally, a prokaryotic expression vector was constructed, and the purified heat shock protein MaltHSP20‑11 of Monochamus alternata was successfully obtained through in vitro expression. to you + The heat shock protein MaltHSP20 of Monochamus alternatus purified by resin column was verified for molecular chaperone activity, which showed that the protein could effectively protect the heat coagulation of malate dehydrogenase. Therefore, this application has extremely high application value, and also provides a molecular basis for the research on the heat resistance of Monochamus alternatus.

Description

technical field [0001] The invention belongs to the field of heat shock proteins, and more particularly, relates to a heat shock protein MaltHSP20-11 of A. pineapple and its encoding gene and application. Background technique [0002] Monochamus alternatus belongs to Coleoptera, family Cerambycidae. It is not only an important stem borer pest in pine forests in southern my country, but also the main transmission vector of pine wood nematode, a quarantine pest in the world. The infection cycle of Longhorn beetle and P. xylophilus destroys the pine conducting tissue, leading to pine wilt disease, which poses a huge threat to the forest resources of my country and the world. Pseudomonas longa is widely distributed in the subtropical and tropical regions of my country. Due to the emergence of global warming, extreme high temperatures frequently occur. The pest needs to experience high temperatures of nearly 40°C in summer and has strong heat resistance. [0003] In the long-ter...

Claims

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Application Information

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Patent Type & Authority Patents(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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