柑橘全爪螨热激蛋白基因PcHsp90的克隆及表达模式分析  

Cloning and expression profiling of heat shock protein gene PcHsp90 in the citrus red mite,Panonychus citri( Acari: Tetranychidae)

作　　者：杨丽红[1,2] 豆威[2] 蒋红波[2] 牛金志[2] 丁天波[2] 王进军[2] 

机构地区：[1]绵阳师范学院生命科学与技术学院,生态安全与保护四川省重点实验室,四川绵阳621006 [2]西南大学植物保护学院,昆虫学及害虫控制工程重庆市市级重点实验室,重庆400715

出　　处：《昆虫学报》2016年第4期411-420,共10页Acta Entomologica Sinica

基　　金：国家公益性行业(农业)科研专项(201103020);绵阳师范学院项目(2012A15,QD2013A06)

摘　　要：【目的】研究热激蛋白基因Hsp90在柑橘全爪螨Panonychus citri生长发育及响应高温和低温胁迫方面的作用。【方法】采用RT-PCR和RACE技术克隆柑橘全爪螨Hsp90基因cDNA全长序列;利用生物信息学软件分析该基因的序列特性;运用荧光Real-time PCR技术,分析Hsp90基因mRNA在该螨各发育阶段、高温及低温胁迫条件下的表达模式。【结果】克隆鉴定出柑橘全爪螨一条Hsp90基因的c DNA全长序列,命名为PcHsp90（Gen Bank登录号：GQ495086）,全长为2 763 bp,包含2 193 bp的开放阅读框,编码730个氨基酸,编码蛋白质的理论分子量和等电点分别为83.85kDa和4.99,氨基酸序列包括Hsp90家族的5个特征基序及细胞质型Hsp90的特征序列＂MEEVD＂。系统进化分析表明,PcHsp90与朱砂叶螨Tetranychus cinnabarinus的Hsp90首先聚为一支,然后再与肩突硬蜱Ixodes scapularis的Hsp90聚合,说明它们较近的亲缘关系。PcHsp90在柑橘全爪螨的各发育阶段均有所表达,其中在幼螨期表达量较低,且显著低于若螨和成螨期的表达水平（P=0.015）。0~10℃低温胁迫下,Pc Hsp90的mRNA相对表达量无显著变化（P=0.492）;但在35~41℃高温胁迫下,Pc Hsp90的mRNA相对表达量随胁迫温度的升高而上调,尤其是当温度升高到41℃时,mRNA相对表达量达到对照（25℃）的6.75倍,且差异达显著水平（P=0.007）。【结论】柑橘全爪螨PcHsp90不仅对该螨的生长发育具有重要作用,而且是其响应高温胁迫的重要机制之一。【Aim】To explore the roles of Hsp90 gene in development and response to heat and cold stress in the citrus red mite,Panonychus citri. 【Methods】The full-length c DNA encoding Hsp90 from P. citri was cloned and identified using RT-PCR and RACE technique. Bioinformatics programs were used to analyze the sequence characteristics of the gene. The mRNA expression levels of the gene in this mite at different developmental stages and under cold and heat stress conditions were detected by real-time PCR.【Results】The complete c DNA of the cloned gene Pc Hsp90 with Gen Bank accession no. GQ495086 is2 763 bp in length,and contains an open reading frame of 2 193 bp,which encodes a protein of 730 amino acids,with the molecular weight of 83. 85 k Da and the theoretical isoelectric point of 4. 99. The deduced amino acid sequence contains five highly conserved motifs of Hsp90 family and the cytoplasmic Hsp90 C-terminal region ＂MEEVD＂. Phylogenetic analysis showed that Pc Hsp90 clustered firstly with Hsp90 of Tetranychus cinnabarinus, and then with that of Ixodes scapularis, suggesting the close relationship among them. Pc Hsp90 transcripts were all detected at different developmental stages,and the relative mRNA expression level was significantly lower at larval stage than those at nymphal and adult stages（ P = 0. 015）. No distinct change was found in relative Pc Hsp90 mRNA expression levels in the mites subjected to 0- 10℃ cold stress（ P = 0. 492）,but a significant up-regulation was observed in the mites subjected to 35- 41℃ heat stress（ P = 0. 007）; especially when the temperature was up to 41℃,the mRNA level was 6. 75-fold as high as that of the control（ 25℃）. 【Conclusion】Pc Hsp90 may be involved in development and plays an important role in response to heat stress in P. citri.

关 键 词：柑橘全爪螨 热激蛋白90 CDNA克隆 低温胁迫 高温胁迫 发育阶段 mRNA表达 

分 类 号：Q966[生物学—昆虫学]