《环境昆虫学报》

作者	单位
杨华磊，李慧，赵培渊，许丹雯仪，李长燕，郝德君	1. 南京林业大学南方现代林业协同创新中心，南京 210037；2. 南京林业大学林学院，南京 210037

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中文摘要:

松墨天牛Monochamus alternatus是我国南方林区重要的蛀干害虫，也是我国重大林业外来入侵物种松材线虫Bursaphelenchus xylophilus的主要传播媒介。为探究热激蛋白HSP40在松墨天牛抵御高温胁迫中的功能，基于松墨天牛转录组数据（GenBank登录号：PRJNA548205），通过RT-PCR技术克隆两条松墨天牛HSP40基因，并对其进行生物信息学分析；使用MEGA 7.0软件构建松墨天牛HSP40系统进化树；利用RT-qPCR技术检测16日龄松墨天牛雌雄成虫各组织HSP40基因在不同温度与时间处理条件（35℃、37℃、40℃、42.5℃和45℃；0 h、1 h、2 h 和3 h）下和高温42.5℃处理3 h后的组织表达特性。结果表明：克隆获得松墨天牛两条HSP40基因MaltHSP40-1（GenBank登录号：MW690168）和MaltHSP40-2（GenBank登录号：MW690169），其ORF长度分别为1 206 bp和1 059 bp，分别编码401个和352个氨基酸，分子质量分别约为45.24 kDa和39.25 kDa，等电点分别为6.73和9.07；两条序列中均存在含有保守的HPD基序以及DNA-J结构域；蛋白三维结构中均由N端的4个α螺旋和C端的底物结合域构成。系统进化树显示：MaltHSP40-1和MaltHSP40-2分别与光肩星天牛Anoplophora glabripennis的AglaHSP40-1和AglaHSP40-2遗传距离最近。RT-qPCR结果显示，高温胁迫可诱导松墨天牛HSP40基因的转录表达，雌雄成虫的MaltHSP40-1和MaltHSP40-2分别在35℃和37.5℃的条件下开始表达上调，在40℃或42.5℃时到达峰值，雄虫HSP40的相对表达量和上调倍数均高于雌虫；MaltHSP40-1及MaltHSP40-2在松墨天牛雌雄成虫的各组织中均有表达，42.5℃处理3 h后，MaltHSP40-1及MaltHSP40-2在各组织中的相对表达量均显著上调，精巢和卵巢中相对表达量最高。本研究表明MaltHSP40-1和MaltHSP40-2作为辅助分子伴侣，参与松墨天牛抵御高温胁迫。研究结果有助于深入探究热激蛋白在松墨天牛应对高温胁迫中的作用，也为揭示气候变暖背景下松墨天牛及松材线虫病的流行成灾机理提供理论依据。

英文摘要:

Monochamus alternatus is a significant stem borer in Southern China, and it is also the primary vector for Bursaphelenchus xylophilus, a major forest invasive species in China. To explore the role of heat shock protein HSP40 in M.alternatus' resistance to high-temperature stress, two HSP40 genes of M. alternatus was cloned using RT-PCR and analyzed by bioinformatics, based on the transcriptome data of M.alternatus(GenBank accession number: PRJNA548205). The phylogenetic tree of M. alternatus HSP40 was constructed using MEGA 7.0 software. RT-qPCR was used to detect the expression of HSP40 gene in different tissues of 16-day-old M. alternatus male and female adults under different temperature and time heat shock treatment conditions (35℃, 37℃, 40℃, 42.5℃ and 45℃; 0, 1 h, 2 h and 3 h) and high temperature treatment at 42.5℃ for 3 h. The findings showed that two HSP40 genes, MaltHSP40-1(GenBank accession number: MW690168) and MaltHSP40-2(GenBank accession number: MW690169), were cloned from M. alternatus. Their ORF lengths were 1 206 bp and 1 059 bp, encoding 401 and 352 amino acids, respectively. The molecular weights were about 45.24 kDa and 39.25 kDa, and the isoelectric points ranged from 6.73 to 9.07. DNA-J containing conserved HPD motif was found in both sequences. The three-dimensional structure of the protein comprises four α-helices at the N-terminus and a substrate binding domain at the C-terminus. The phylogenetic tree showed that MaltHSP40-1 and MaltHSP40-2 had the closest genetic distance with AglaHSP40-1 and AglaHSP40-2 of Anoplophora glabripennis, respectively. The results of RT-qPCR showed that high temperature stress could induce the expression of HSP40 in M.alternatus. The expression of MaltHSP40-1 and MaltHSP40-2 in male and female adults began to increase at 35℃ and 37.5℃, and reached the peak at 40℃ or 42.5℃. The relative expression and up-regulation of HSP40 in males were higher than those in females. Additionally, MaltHSP40-1 and MaltHSP40-2 were expressed in all tissues of male and female adults of M.alternatus. After treatment at 42.5℃ for 3 h, the relative expression levels of MaltHSP40-1 and MaltHSP40-2 in all tissues significantly increased, with the highest relative expression in testes and ovaries. These results could further facilitate an exploration of the role of heat shock proteins in M.alternatus' response to high-temperature stress. They also provide a theoretical basis for revealing the epidemic mechanism of M. alternatus and B. xylophilus disease under the background of climate warming.

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