用荧光定量RT-PCR方法分析B型和ZHJ-1型烟粉虱热休克基因hsp70和hsp90的表达  

Detection of hsp70 and hsp90 Transcription in Bemisia tabaci (B and ZHJ-1 Biotype) by Fluorescence Quantitative RT-PCR

作　　者：王海鸿[1] 雷仲仁[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100094

出　　处：《中国农业科学》2008年第11期3636-3644,共9页Scientia Agricultura Sinica

基　　金：国家"973"项目(2002CB111402和2009CB119000)

摘　　要：【目的】比较温度胁迫对两种生物型烟粉虱体内hsps诱导表达的差异。【方法】根据烟粉虱hsp70基因、hsp90基因和18Sr RNA序列,设计并合成引物用于实时定量RT-PCR,扩增产物连接到质粒pMD18-T载体上,重组质粒经筛选、签定和10×梯度稀释后作为模板,用于标准曲线的制作和样品检测,检测温度胁迫对B型和ZHJ-1型烟粉虱hsp70和hsp90mRNA转录水平的诱导情况。【结果】在受到34℃、37℃、40℃和43℃的高温胁迫时,两者hsps的诱导水平无显著差异(P>0.05);在受到4℃、7℃、10℃和13℃的低温胁迫时,B型烟粉虱体内hsps的诱导表达水平显著超过了ZHJ-1型烟粉虱(P<0.05)。【结论】经检验所建立的荧光定量RT-PCR体系特异性好,灵敏度高,符合检测烟粉虱hsps mRNA转录水平的要求。低温胁迫下B型烟粉虱体内hsps的高表达可能是其较强的耐受低温能力的主要机制之一。[Objective] To study the transcription difference of the hsps between biotype B and ZHJ-1 of whitefly （Bemisia tabaci）. [Method] Primers were dsigned according to the specific sequence hsp70 gene, hsp90 gene and 18S rRNA of whiteflies, and they were used in the fluorescence quantitative RT-PCR （FQ RT-PCR）. The fragments generated by PCR were cloned into the pMD18-T vector. After being screened, confirmed and diluted, the positive recombinant plasmids were used as standard quantitative templates to make the standard curve and detect the samples. In this FQ RT-PCR system, hsps transcription of biotypes B and ZHJ-1 of whiteflies induced by stress temperature was compared. [ Result ] Under elevated temperatures（34℃, 37 ℃, 40℃ and 43 ℃）, the two biotypes had no significant difference in induction levels ofhsps （P〉0.05）. The transcription levels ofhsps mRNA in biotype B were significantly higher （P〈0.05） than those of biotype ZHJ-1 when they underwent cold stress（at 4℃, 7℃, 10℃ and 13℃）. [Conclusion] The fluorescence quantitative RT-PCR （FQ RT-PCR） method established is qualified after the examination.The overexpressive hsps mRNA of biotype B under depressed temperatures may be related to its tolerance of low temperature.

关 键 词：烟粉虱 热休克基因 SYBRGreenI 荧光定量RT-PCR 温度胁迫 

分 类 号：S433[农业科学—农业昆虫与害虫防治]