松墨天牛GST基因克隆及高温胁迫下的表达特性分析

李子纯; 郝德君; 李慧; 李长燕; 许丹雯仪; 杨华磊; 赵培渊

doi:10.12302/j.issn.1000-2006.202304018

松墨天牛GST基因克隆及高温胁迫下的表达特性分析

李子纯, 郝德君, 李慧, 李长燕, 许丹雯仪, 杨华磊, 赵培渊

南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1) : 28-36.

PDF(13858 KB)

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

PDF(13858 KB)

南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (1) : 28-36. DOI: 10.12302/j.issn.1000-2006.202304018

专题报道：松材线虫病绿色防控研究（执行主编叶建仁骆有庆）

松墨天牛GST基因克隆及高温胁迫下的表达特性分析

作者信息 +

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

Author information +

文章历史 +

摘要

【目的】克隆松墨天牛(Monochamus alternatus)谷胱甘肽S转移酶(glutathione S-transferases, GST)相关基因,明确GST基因在松墨天牛响应高温胁迫中的效用,为探究亚热带地区松墨天牛的耐热分子机制提供理论依据。【方法】克隆3条松墨天牛GST基因,结合DNAMAN 9.0、I-TASSER等软件分析松墨天牛GST基因的结构特征;利用qRT-PCR技术测定分析松墨天牛成虫和4龄幼虫在不同高温、不同处理时长后GST基因的相对表达量;通过纸盘扩散法验证3条松墨天牛GST基因在保护机体免受氧化应激中的作用。【结果】克隆3条松墨天牛GST基因的cDNA序列,分别命名为MaltGSTe1、MaltGSTe2和MaltGSTt1。MaltGSTe1与MaltGSTe2均属于GST的Epsilon家族,MaltGSTt1属于GST的Theta家族。3条GST基因三维蛋白结构具有指示性结构特征,属于胞质型GST。松墨天牛4龄幼虫在高温胁迫下MaltGSTe1、MaltGSTe2和MaltGSTt1的相对表达量均出现显著变化,MaltGSTe2相对表达水平上调幅度最大;MaltGSTt1在松墨天牛雄虫体内相对表达量出现明显下调;异源表达3条GST基因蛋白的大肠杆菌表现出较强的抗氧化能力,其中,MaltGSTe2具有更强的抗氧化能力。【结论】克隆获得3条松墨天牛GST基因,发现高温胁迫可诱导GST基因表达量上调;纸盘扩散分析结果表明异源表达GST基因蛋白具有抗氧化能力,推测GST基因具有通过保护机体免受氧化应激来参与松墨天牛幼虫的高温胁迫响应机制。

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.

导出引用

李子纯, 郝德君, 李慧, 等. 松墨天牛GST基因克隆及高温胁迫下的表达特性分析[J]. 南京林业大学学报（自然科学版）. 2025, 49(1): 28-36 https://doi.org/10.12302/j.issn.1000-2006.202304018

LI Zichun, HAO Dejun, LI Hui, et al. 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 https://doi.org/10.12302/j.issn.1000-2006.202304018

中图分类号： S763.38

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	GARRAD R, BOOTH D T, FURLONG M J. The effect of rearing temperature on development, body size, energetics and fecundity of the diamondback moth[J]. Bull Entomol Res, 2016, 106(2): 175-181. DOI: 10.1017/S000748531500098X. 本文引用 [1]

[2]

钱雪, 王月莹, 谢欢欢, 等. 温度对西伯利亚蝗呼吸代谢关键酶活性的影响[J]. 昆虫学报, 2017, 60(5): 499-504.

QIAN

, WANG

Y Y

, XIE

H H

, et al. Effects of temperature on the activities of key enzymes related to respiratory metabolism in adults of Gomphocerus sibiricus (Orthoptera: Acrididae)[J]. Acta Entomol Sin, 2017, 60(5): 499-504. DOI: 10.16380/j.kcxb.2017.05.001.

本文引用 [1]

[3]	TURRENS J F. Mitochondrial formation of reactive oxygen species[J]. J Physiol, 2003, 552(2): 335-344. DOI: 10.1113/jphysiol.2003.049478. 本文引用 [1]

[4]	HALLIWELL B. Antioxidants: the basics, what they are and how to evaluate them[J]. Adv Pharmacol, 1996, 38: 3-20. DOI: 10.1016/s1054-3589(08)60976-x. 本文引用 [1]

[5]	SLIMEN I B, NAJAR T, GHRAM A, et al. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage: a review[J]. Int J Hyperthermia, 2014, 30(7): 513-523. DOI: 10.3109/02656736.2014.971446. 本文引用 [1]

[6]	HABASHY W S, MILFORT M C, REKAYA R, et al. Cellular antioxidant enzyme activity and biomarkers for oxidative stress are affected by heat stress[J]. Int J Biometeorol, 2019, 63(12): 1569-1584. DOI: 10.1007/s00484-019-01769-z. 本文引用 [1]

[7]

李慧, 郝德君, 徐天, 等. 高温胁迫对植食性昆虫影响研究进展[J]. 南京林业大学学报(自然科学版), 2022, 46(6): 215-224.

, HAO

D J

, XU

, et al. The effects of heat stress on herbivorous insects: an overview and future directions[J]. J Nanjing For Univ (Nat Sci Ed), 2022, 46(6): 215-224. DOI: 10.12302/j.issn.1000-2006.202209041.

本文引用 [1]

[8]	RIX R R, CUTLER G C. Review of molecular and biochemical responses during stress induced stimulation and hormesis in insects[J]. Sci Total Environ, 2022, 827: 154085. DOI: 10.1016/j.scitotenv.2022.154085. 本文引用 [1]

[9]	ZHANG S Z, FU W Y, LI N, et al. Antioxidant responses of Propylaea japonica (Coleoptera: Coccinellidae) exposed to high temperature stress[J]. J Insect Physiol, 2015, 73: 47-52. DOI: 10.1016/j.jinsphys.2015.01.004. 本文引用 [1]

[10]	KANG Z W, LIU F H, LIU X, et al. The potential coordination of the heat-shock proteins and antioxidant enzyme genes of Aphidius gifuensis in response to thermal stress[J]. Front Physiol, 2017, 8: 976. DOI: 10.3389/fphys.2017.00976. 本文引用 [2]

[11]	MOREIRA D C, PAULA D P, HERMES-LIMA M. Changes in metabolism and antioxidant systems during tropical diapause in the sunflower caterpillar Chlosyne lacinia (Lepidoptera: Nymphalidae)[J]. Insect Biochem Mol Biol, 2021, 134: 103581. DOI: 10.1016/j.ibmb.2021.103581. 本文引用 [1]

[12]	WANG Y J, QIU L, RANSON H, et al. Structure of an insect epsilon class glutathione S-transferase from the malaria vector Anopheles gambiae provides an explanation for the high DDT-detoxifying activity[J]. J Struct Biol, 2008, 164(2): 228-235. DOI: 10.1016/j.jsb.2008.08.003. 本文引用 [1]

[13]	OAKLEY A. Glutathione transferases: a structural perspective[J]. Drug Metab Rev, 2011, 43(2): 138-151. DOI: 10.3109/03602532.2011.558093. 本文引用 [1]

[14]	MANNERVIK B. Five decades with glutathione and the GSTome[J]. J Biol Chem, 2012, 287(9): 6072-6083. DOI: 10.1074/jbc.X112.342675. 本文引用 [1]

[15]	GALLÉ Á, BELA K, HAJNAL Á, et al. Crosstalk between the redox signalling and the detoxification:GSTs under redox control?[J]. Plant Physiol Biochem, 2021, 169: 149-159. DOI: 10.1016/j.plaphy.2021.11.009. 本文引用 [1]

[16]	HAYES J D, FLANAGAN J U, JOWSEY I R. Glutathione transferases[J]. Annu Rev Pharmacol Toxicol, 2005, 45: 51-88. DOI: 10.1146/annurev.pharmtox.45.120403.095857. 本文引用 [1]

[17]	RAZA H. Dual localization of glutathione S-transferase in the cytosol and mitochondria: implications in oxidative stress, toxicity and disease[J]. FEBS J, 2011, 278(22): 4243-4251. DOI: 10.1111/j.1742-4658.2011.08358.x. 本文引用 [1]

[18]	LABORDE E. Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death[J]. Cell Death Differ, 2010, 17(9): 1373-1380. DOI: 10.1038/cdd.2010.80. 本文引用 [1]

[19]	ZHU G D, XUE M, LUO Y, et al. Effects of short-term heat shock and physiological responses to heat stress in two Bradysia adults, Bradysia odoriphaga and Bradysia difformis[J]. Sci Rep, 2017, 7(1): 13381. DOI: 10.1038/s41598-017-13560-4. 本文引用 [1]

[20]	ZHAO Y, LI Y Y, HE M, et al. Antioxidant responses of the pest natural enemy Hylyphantes graminicola (Araneae: Linyphiidae) exposed to short-term heat stress[J]. J Therm Biol, 2020, 87: 102477. DOI: 10.1016/j.jtherbio.2019.102477. 本文引用 [1]

[21]	ALVES M, PEREIRA A, MATOS P, et al. Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus[J]. Sci Rep, 2016, 6: 23908. DOI: 10.1038/srep23908. 本文引用 [1]

[22]	TOGASHI K. Population density of Monochamus alternatus adults (Coleoptera: Cerambycidae) and incidence of pine wilt disease caused by Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)[J]. Res Popul Ecol, 1988, 30(2): 177-192. DOI: 10.1007/BF02513243. 本文引用 [1]

[23]

王立超, 陈凤毛, 董晓燕, 等. 松墨天牛取食和产卵特性研究[J]. 南京林业大学学报(自然科学版), 2023, 47(2): 219-224.

WANG

L C

, CHEN

F M

, DONG

X Y

, et al. A study on feeding and oviposition characteristics of Monochamus alternatus[J]. J Nanjing For Univ (Nat Sci Ed), 2023, 47(2): 219-224. DOI: 10.12302/j.issn.1000-2006.202103022.

本文引用 [1]

[24]

陈宏健, 郝德君, 田敏, 等. 室内饲养松墨天牛幼虫不同肠段细菌的群落结构及功能分析[J]. 南京林业大学学报(自然科学版), 2021, 45(3): 143-151.

CHEN

H J

, HAO

D J

, TIAN

, et al. The community structure and functional analysis of intestinal bacteria in Monochamus alternatus larvae reared indoors[J]. J Nanjing For Univ (Nat Sci Ed), 2021, 45(3): 143-151. DOI: 10.12302/j.issn.1000-2006.202004009.

本文引用 [1]

[25]	田开慧, 陈怡帆, 周宏威. 湖南湘西州马尾松毛虫和松材线虫病发生非线性建模与预测[J]. 森林工程, 2022, 38 (6): 38-44. TIAN K H, CHEN Y F, ZHOU H W. Prediction of occurrence trend of Dendrolimus punctatus and pine wilt disease in Xiangxi Prefecture[J]. For Eng, 2022, 38 (6): 38-44. 本文引用 [1]

[26]

王洋, 陈军, 陈凤毛, 等. 松墨天牛取食期间传播松材线虫的特性[J]. 南京林业大学学报(自然科学版), 2019, 43(6): 1-10.

WANG

, CHEN

F M

, et al. Transmission of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) through feeding activity of Monochamus alternatus (Coleoptera: Cerambycidae)[J]. J Nanjing For Univ (Nat Sci Ed), 2019, 43(6): 1-10. DOI: 10.3969/j.issn.1000-2006.201903001.

本文引用 [1]

[27]	ZHAO L L, ZHANG S, WEI W, et al. Chemical signals synchronize the life cycles of a plant-parasitic nematode and its vector beetle[J]. Curr Biol, 2013, 23(20): 2038-2043. DOI: 10.1016/j.cub.2013.08.041. 本文引用 [1]

[28]

OHSAWA

, AKIBA

. Possible altitude and temperature limits on pine wilt disease: the reproduction of vector sawyer beetles(Monochamus alternatus), survival of causal nematode (Bursaphelenchus xylophilus), and occurrence of damage caused by the disease[J]. Eur J For Res, 2014, 133(2): 225-233. DOI: 10.1007/s10342-013-0742-x.

本文引用 [1]

[29]

吴佳雯, 尹艳楠, 谈家金, 等. 蜡样芽孢杆菌NJSZ-13菌株诱导马尾松抗松材线虫病研究[J]. 南京林业大学学报(自然科学版), 2022, 46(4): 53-58.

J W

, YIN

Y N

, TAN

J J

, et al. A preliminary study on resistance of Pinus massoniana induced by Bacillus cereus NJSZ-13 strain to Bursaphelenchus xylophilus[J]. J Nanjing For Univ (Nat Sci Ed), 2022, 46(4): 53-58. DOI: 10.12302/j.issn.1000-2006.202006051.

本文引用 [1]

[30]	杨帆, 零雅茗, 舒红, 等. 松材线虫Bx-TIMP克隆及功能研究[J]. 森林工程, 2022, 38 (2): 14-19. YANG F, LING Y M, SHU H, et al. Cloning and functional analysis of Bursaphelenchus xylophilus Bx-TIMP[J]. For Eng, 2022, 38 (2): 14-19. 本文引用 [1]

[31]

李慧, 何玄玉, 陶蓉, 等. 松墨天牛小热激蛋白基因的克隆、表达谱及对温度胁迫的响应[J]. 昆虫学报, 2018, 61(7): 749-760.

, HE

X Y

, TAO

, et al. cDNA cloning and expression profiling of small heat shock protein genes and their response to temperature stress in Monochamus alternatus(Coleoptera: Cerambycidae)[J]. Acta Entomol Sin, 2018, 61(7): 749-760. DOI: 10.16380/j.kcxb.2018.07.001.

本文引用 [1]

[32]	LI H, QIAO H, LIU Y J, et al. Characterization, expression profiling, and thermal tolerance analysis of heat shock protein 70 in pine sawyer beetle,Monochamus alternatus Hope (Coleoptera: Cerambycidae)[J]. Bull Entomol Res, 2021, 111(2): 217-228. DOI: 10.1017/S0007485320000541. 本文引用 [1]

[33]	CAI Z L, CHEN J X, CHENG J, et al. Overexpression of three heat shock proteins protects Monochamus alternatus (Coleoptera: Cerambycidae) from thermal stress[J]. J Insect Sci, 2017, 17(6): 113. DOI: 10.1093/jisesa/iex082. 本文引用 [1]

[34]	李慧. 热激蛋白在松墨天牛响应高温胁迫中的功能研究[D]. 南京: 南京林业大学, 2021. LI H. Function analysis of heat shock protein in Monochamus alternatus response to high temperature[D]. Nanjing: Nanjing For Univ, 2021. DOI: 10.27242/d.cnki.gnjlu.2021.000015. 本文引用 [3]

[35]	LI H, ZHAO X Y, QIAO H, et al. Comparative transcriptome analysis of the heat stress response in Monochamus alternatus Hope (Coleoptera: Cerambycidae)[J]. Front Physiol, 2020, 10: 1568. DOI: 10.3389/fphys.2019.01568. 本文引用 [2]

[36]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR[J]. Methods, 2002, 25(4): 402-408. DOI: 10.1006/meth.2001.1262. 本文引用 [2]

[37]	RAJARAPU S P, MAMIDALA P, HERMS D A, et al. Antioxidant genes of the emerald ash borer(Agrilus planipennis): gene characterization and expression profiles[J]. J Insect Physiol, 2011, 57(6): 819-824. DOI: 10.1016/j.jinsphys.2011.03.017. 本文引用 [2]

[38]

SANDAMALIKA

W M G

, PRIYATHILAKA

T T

, LEE

, et al. Immune and xenobiotic responses of glutathione S-Transferase theta (GST-θ) from marine invertebrate disk abalone (Haliotis discus discus): with molecular characterization and functional analysis[J]. Fish Shellfish Immunol, 2019, 91: 159-171. DOI: 10.1016/j.fsi.2019.04.004.

本文引用 [2]

[39]	SUN L L, YIN J J, DU H, et al. Characterisation of GST genes from the Hyphantria cunea and their response to the oxidative stress caused by the infection of Hyphantria cunea nucleopolyhedrovirus (HcNPV)[J]. Pestic Biochem Physiol, 2020, 163: 254-262. DOI: 10.1016/j.pestbp.2019.11.019. 本文引用 [1]

[40]

李长春, 宁青, 戴余军, 等. 拟环纹豹蛛谷胱甘肽S-转移酶基因的克隆及表达分析[J]. 江苏农业学报, 2019, 35(5): 1068-1074.

C C

, NING

, DAI

Y J

, et al. Cloning and expression analysis of glutathione S-transferase gene in Pardosa pseudoannulata[J]. Jiangsu J Agri Sci, 2019, 35(5): 1068-1074. DOI: 10.3969/j.issn.1000-4440.2019.05.010.

本文引用 [1]

[41]	YANG Q, LIU J P, WYCKHUYS K A G, et al. Impact of heat stress on the predatory ladybugs Hippodamia variegata and Propylaea quatuordecimpunctata[J]. Insects, 2022, 13(3): 306. DOI: 10.3390/insects13030306. 本文引用 [1]

[42]	YANG X J, ZHENG H L, LIU Y Y, et al. Selection of reference genes for quantitative real-time PCR in Aquatica leii (Coleoptera: Lampyridae) under five different experimental conditions[J]. Front Physiol, 2020, 11: 555233. DOI: 10.3389/fphys.2020.555233. 本文引用 [1]

[43]	张媛英. 中华蜜蜂谷胱甘肽S-转移酶和小分子热激蛋白基因的生物学功能分析[D]. 泰安: 山东农业大学, 2014. ZHANG Y Y. Biological analysis of glutathione S-transferase and small heat shock protein genes in Apis cerana cerana[D]. Tai’an: Shandong Agricul Univ, 2014. DOI: 10.7666/d.Y2587648. 本文引用 [1]