沙葱萤叶甲丝氨酸蛋白酶基因GdSP的克隆及对温度胁迫的响应  

Molecular cloning of a serine protease gene DdSP and its response to temperature stress in Galeruca daurica( Coleoptera: Chrysomelidae)

作　　者：单艳敏 张玉 霍志家 庞保平 孙学涛 SHAN Yan-Min;ZHANG Yu;HUO Zhi-Jia;PANG Bao-Ping;SUN Xue-Tao(Research Center for Grassland Entomology,Inner Mongolia Agricultural University,Hohhot 010010,China;Grassland Station of Inner Mongolia Autonomous Region,Hohhot 010020,China;Grassland Station of Chifeng City,Chifeng,Inner Mongolia 024000,China)

机构地区：[1]内蒙古农业大学草原昆虫研究中心,呼和浩特010010 [2]内蒙古自治区草原工作站,呼和浩特010020 [3]赤峰市草原工作站,内蒙古赤峰024000

出　　处：《昆虫学报》2018年第7期761-770,共10页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31360441)

摘　　要：【目的】丝氨酸蛋白酶(serine protease,SP)是一类以丝氨酸为活性中心的重要的蛋白水解酶。本研究旨在克隆获得沙葱萤叶甲Galeruca daurica丝氨酸蛋白酶基因,分析其对温度胁迫的响应,以期为进一步揭示沙葱萤叶甲耐温性的调控机制及其他生理功能奠定基础。【方法】根据沙葱萤叶甲2龄幼虫转录组数据,采用RACE技术克隆得到沙葱萤叶甲丝氨酸蛋白酶基因的cDNA全长序列,并进行生物信息学分析;应用qPCR技术检测其在不同温度(-10,-5,0,5,25和35℃)下处理1 h后及25℃下恢复30 min后在沙葱萤叶甲2龄幼虫中的表达量变化。【结果】自沙葱萤叶甲克隆获得一个丝氨酸蛋白酶基因,命名为GdSP(GenBank登录号:MG797556)。该基因全长1 110 bp,开放阅读框969 bp,编码322个氨基酸;蛋白预测分子量35.41 kD,等电点5.61;编码蛋白具有丝氨酸蛋白酶的典型特征,具有一个跨膜结构,无信号肽。同源序列比对和系统发育分析表明,GdSP与光肩星天牛Anoplophora glabripennis SP的同源性最高,氨基酸序列一致性为30.53%。qPCR测定结果表明,不同温度处理间2龄幼虫中GdSP表达量差异不显著,但对各高低温(-10℃除外)胁迫处理回温后GdSP表达量显著上升。【结论】快速冷驯化对沙葱萤叶甲丝氨酸蛋白酶基因表达无显著影响,而回温可诱导其上调表达。【Aim】Serine protease(SP)is an important proteolytic enzyme,with serine as its active center.This study aims to clone a serine protease gene from Galeruca daurica and to analyze its expression levels in response to temperature stress so as to lay a foundation for further investigation on the regulation mechanisms of temperature tolerance and other physiological functions in G.daurica.【Methods】Based on the transcriptome data of the 2nd instar larvae of G.daurica,the full-length cDNA of the serine protease gene was cloned from G.daurica by RACE,and its sequence was subjected to bioinformatics analysis.Its expression profiles in the 2nd instar larvae of G.daurica exposed to different temperatures(-10,-5,0,5,25,and 35℃)for 1 h and recovered at 25℃for 30 min were detected by qPCR.【Results】A serine protease gene was cloned from G.daurica,and named GdSP(GenBank accession no.:MG797556).GdSP is 1 110 bp in length with an open reading frame(ORF)of 969 bp,encoding a protein of 322 amino acids with the predicted molecular weight of 35.41 kD and pI of 5.61.The encoded protein shares the typical structural features of serine proteases,with a transmembrane domain but no signal peptide.Homologous sequence alignment and phylogenetic analysis showed that GdSP has the highest amino acid sequence identity(30.53%)with Anoplophora glabripennis SP.qPCR results showed that the expression levels of GdSP had no significant difference in the 2nd instar larvae of G.daurica exposed to different temperatures whereas increased significantly after recovery from the low(except-10℃)and high temperature stresses.【Conclusion】Rapid cold-hardening has no significant effects on the expression of GdSP while recovery from cold shock elicits its up-regulated expression.

关 键 词：沙葱萤叶甲 丝氨酸蛋白酶 基因克隆 温度胁迫 表达分析 

分 类 号：Q966[生物学—昆虫学]