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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 被引量:9

Detection of hsp70 and hsp90 Transcription in Bemisia tabaci (B and ZHJ-1 Biotype) by Fluorescence Quantitative RT-PCR
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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.
出处 《中国农业科学》 CAS CSCD 北大核心 2008年第11期3636-3644,共9页 Scientia Agricultura Sinica
基金 国家"973"项目(2002CB111402和2009CB119000)
关键词 烟粉虱 热休克基因 SYBRGreenI 荧光定量RT-PCR 温度胁迫 Bemisia tabaci Heat shock gene SYBR Green I FQ RT-PCR Temperature stress
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参考文献39

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