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.pdfVIP

中国农业科学 2008,41(11):3636-3644 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2008.11.025 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 王海鸿，雷仲仁 （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 ） 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18S rRNA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 hsp90 mRNA 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。 关键词：烟粉虱；热休克基因；SYBR Green I；荧光定量RT-PCR；温度胁迫 Detection of hsp70 and hsp90 Transcription in Bemisia tabaci (B and ZHJ-1 Biotype) by Fluorescence Quantitative RT-PCR WANG Hai-hong, LEI Zhong-ren (State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094) Abstract: 【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 stre