Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress

doi:10.12302/j.issn.1000-2006.202304018

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2025, Vol. 49 ›› Issue (1): 28-36.doi: 10.12302/j.issn.1000-2006.202304018

Special Issue: 专题报道：松材线虫病绿色防控研究

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Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress

LI Zichun(), HAO Dejun^*(), LI Hui, LI Changyan, XU Danwenyi, YANG Hualei, ZHAO Peiyuan

Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China

Received:2023-04-10 Revised:2024-02-02 Online:2025-01-30 Published:2025-01-21
Contact: HAO Dejun E-mail:leezichun@njfu.edu.cn;dejunhao@163.com

Abstract

Abstract:

【Objective】To clone the glutathione S-transferases (GST) genes of Monochamus alternatus (Coleoptera: Cerambycidae) and to determine the expression levels of these genes under heat stress in adult males and females, as well as at different developmental stages of M. alternatus, we used molecular biology and bioinformatics methods. 【Method】 Three GST cDNA sequences were cloned, and their structural characteristics were analyzed using DNAMAN 9.0 and I-TASSER software. qRT-PCR was employed to measure the relative expression levels of GST genes in adult and fourth instar larvae of M. alternatus under varying temperatures and treatment durations. The role of these GST genes in protecting the organism from oxidative stress was assessed through disc diffusion assays. 【Result】Three GST cDNA sequences were cloned and named MaltGSTe1, MaltGSTe2, and MaltGSTt1. MaltGSTe1 and MaltGSTe2 belong to the Epsilon family, while MaltGSTt1 belongs to the Theta family. Analysis of three-dimensional protein structures indicated that these GST are cytoplasmic. The relative expression levels of MaltGSTe1, MaltGSTe2 and MaltGSTt1 in fourth instar larvae significantly changed under heat stress, with MaltGSTe2 showing the greatest change. In males, the expression level of MaltGSTt1 was significantly down-regulated. Escherichia coli expressing the GST genes demonstrated enhanced antioxidant capacity, with MaltGSTe2 exhibiting the strongest activity. 【Conclusion】We successfully cloned three GST genes and investigated their expression characteristics under high temperature stress in M. alternatus. High temperature stress was found to induce up-regulation of GST gene expression. Disc diffusion assays confirmed that heterogeneously expressed GST proteins have antioxidant capabilities. These findings suggest that GST genes play a role in the response mechanism to high temperature stress by protecting against oxidative stress, providing a theoretical foundation for exploring the heat resistance mechanisms of M. alternatus in subtropical regions.

Key words: Monochamus alternatus, heat stress, glutathione S-transferases, GST gene, oxidative stress, gene clone, express analysis, bioinformatics

CLC Number:

S763.38

LI Zichun, HAO Dejun, LI Hui, LI Changyan, XU Danwenyi, YANG Hualei, ZHAO Peiyuan. Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2025, 49(1): 28-36.

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引物名称 primer name	引物序列(5'-3') primer sequence	相关系数(R²) correlation coefficient	扩增效率/% amplification efficiency
GAPDH-qF	TCGAACGCTTCATGCACAAC	0.99	1.09
GAPDH-qR	CCATCACGCCACAAATTTTCC
MaltGSTe-1-qF	AACGTACTAGTGCCCATCGC	0.99	1.10
MaltGSTe-1-qR	AGCCTACTTGATTGGCCTCG
MaltGSTe-2-qF	ATACCAGCCCTGGACGACAA	0.99	1.03
MaltGSTe-2-qR	GCTTTTCAACATCGGCAGGA
MaltGSTt-1-qF	ATATGCTGCCGGAGATCACG	1.00	1.09
MaltGSTt-1-qR	ACCAGCTTTGGACAAGAGGAT

引物名称 primer name	引物序列(5'-3') primer sequence
MaltGSTe-1-F^①	ATGGCGCCTAAATTACACTACG
MaltGSTe-1-R^①	TCAACCAAGCTTGCTCTTGAC
MaltGSTe-2-F^①	ATGGCTCCAAAGTTATATATGACA
MaltGSTe-2-R^①	TTACGTGGATAATGCATTTTGAACT
MaltGSTt-1-F^①	ATGCCACTAACATTATATGCTGT
MaltGSTt-1-R^①	TTATTTTCTAATGGGGTGAATGGGA
MaltGSTe-1-tF^②	cagcaaatgggtcgcggatccATGGCGCCTAAATTACACTACGC
MaltGSTe-1-tR^②	ctcgagtgcggccgcaagcttACCAAGCTTGCTCTTGACCATT
MaltGSTe-2-tF^②	cagcaaatgggtcgcggatccATGGCTCCAAAGTTATATATGACAATAA
MaltGSTe-2-tR^②	ctcgagtgcggccgcaagcttCGTGGATAATGCATTTTGAACTAGT
MaltGSTt-1-tF^②	cagcaaatgggtcgcggatccATGCCACTAACATTATATGCTGTATCTG
MaltGSTt-1-tR^②	ctcgagtgcggccgcaagcttTTTTCTAATGGGGTGAATGGGA

基因名称 gene name	登录号 genbank No.	氨基酸数量 amount of amino acids	相对分子质量 relative molecular weight	等电点 isoelectric point
MaltGSTe1	OQ476235	216	23.67	5.94
MaltGSTe2	OQ476236	216	24.05	5.73
MaltGSTt1	OQ476237	230	26.66	5.84

Cloning of glutathione S-transferases gene from Monochamus alternatus and its expression characteristics under heat stress

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