三叶草斑潜蝇hsp70的克隆及其表达量在高低温胁迫下的变化

Cloning of heat shock protein gene,hsp70,in Liriomyza trifolii and its expression under temperature stress

为了明确高低温胁迫对三叶草斑潜蝇hsp70表达量的影响,采用RT-PCR和RACE技术获得了1条三叶草斑潜蝇诱导型热激蛋白基因hsp70,命名为Lthsp70-1,并利用实时荧光定量PCR技术检测其在温度胁迫后的表达量。该基因的开放阅读框为1923bp,编码640个氨基酸。氨基酸序列中含有HSP70家族的签名序列IFDLGGGTFDVSIL和IVLVGGSTRIPK、DnaK特征基序IDLGTT(Y)S(C)V、非细胞器基序RARFEEL,以及C末端的保守序列EEVD。实时荧光定量PCR结果显示:成虫在31～33℃范围内,其hsp70表达量随温度升高而上升,33℃时达到最高峰;35～39℃时,hsp70表达量迅速下降;蛹经0℃胁迫0.5～2.0h,其hsp70表达量随时间延长呈上升趋势。由此可见,高低温胁迫均能诱导三叶草斑潜蝇hsp70的表达。

The objectives of this study were to analyze the effect of temperature stress on the expression of hsp70. The hsp70 in Liriomyza trifolii was cloned using RT-PCR and RACE, and its expression in response to temperature stress was examined using real-time fluorescence quantitative PCR method. A hsp70 gene, named Lthsp70-1, from L. trifolii was cloned and sequenced, the cDNA contains a 1 923 bp open reading frame （ORF） and encodes 640 amino acids. The gene contains the signature sequences of HSP70 family （ IFDLGGGTFDVSIL, IVLVGGSTRIPK）, the DnaK sequence （ IDLGTY （ Y ） S （ C ） V ）, the non-organellar motif （RARFEEL）, and the C-terminal four amino acids of HSP70 （EEVD）. Realtime fluorescence quantitative PCR analysis showed that temperature stress induced the expression of hsp70： the level of expression in adults increased from 31 to 33 ℃, peaked at 33 ℃, and decreased rapidly from 35 to 39 ℃. Exposure of pupa to 0 ℃ for 0.5 to 2.0 h also elicited the expression of hsp70 and the expression level increased with the exposure time. The hsp70 in L. trifolii is inducible, as its expression level varies in response to both low and high temperature stress.