本文關(guān)鍵詞： 馬鈴薯甲蟲 熱激蛋白 HSP 基因克隆 溫度脅迫 實(shí)時熒光定量PCR　出處：《昆蟲學(xué)報(bào)》2017年05期 　論文類型：期刊論文

【摘要】：【目的】馬鈴薯甲蟲Leptinotarsa decemlineata是一種世界性檢疫害蟲,對溫度脅迫具有極強(qiáng)的適應(yīng)性,為進(jìn)一步明確其對溫度脅迫適應(yīng)性的分子機(jī)制,研究了熱激蛋白HSP60在馬鈴薯甲蟲溫度脅迫應(yīng)答過程中的作用。【方法】采用RT-PCR及RACE技術(shù)克隆馬鈴薯甲蟲熱激蛋白HSP60基因的cDNA全長序列;利用生物信息學(xué)軟件分析該基因及其編碼蛋白質(zhì)的序列特性;運(yùn)用實(shí)時熒光定量PCR技術(shù)分析該基因在溫度脅迫下的表達(dá)模式�！窘Y(jié)果】克隆得到馬鈴薯甲蟲熱激蛋白HSP60基因,命名為Ld-HSP60(Gen Bank登錄號:KC556801),其cDNA全長2 234 bp,開放閱讀框(ORF)長1 731 bp,編碼576個氨基酸,相對分子量約為61.27 kD,理論等電點(diǎn)為5.51,5'端非翻譯區(qū)(UTR)長101 bp,3'UTR長402 bp。氨基酸序列中含有HSP60家族典型的特征序列。實(shí)時熒光定量PCR結(jié)果表明,低溫脅迫(-10和0℃)下未檢測到馬鈴薯甲蟲雌雄成蟲中Ld-HSP60的誘導(dǎo)表達(dá);高溫脅迫(38和44℃)誘導(dǎo)馬鈴薯甲蟲雄成蟲Ld-HSP60上調(diào)表達(dá),隨著脅迫溫度的升高LdHSP60表達(dá)量呈現(xiàn)先升高后降低的趨勢,38℃高溫脅迫下表達(dá)量最高,脅迫時間越長Ld-HSP60表達(dá)量也越高。【結(jié)論】相比其他熱激蛋白,HSP60對溫度敏感性較低,推測HSP60可能在馬鈴薯甲蟲雄成蟲抵御高溫脅迫中發(fā)揮作用。
[Abstract]:[objective] Potato beetle Leptinotarsa decemlineata is a worldwide quarantine pest with strong adaptability to temperature stress. In order to further clarify its adaptability to temperature stress molecular mechanism. The effect of heat shock protein (HSP60) on the response of potato beetles to temperature stress was studied. [methods] RT-PCR and RACE techniques were used to clone the c of HSP60 gene of potato beetle. DNA full-length sequence; The sequence characteristics of the gene and its encoded protein were analyzed by bioinformatics software. The expression pattern of this gene under temperature stress was analyzed by real-time fluorescence quantitative PCR. [results] the HSP60 gene of potato beetle heat shock protein was cloned. It was named Ld-HSP60(Gen Bank login number: KC556801.The length of cDNA was 2 234 BP, and the open reading frame (ORF) was 1 731bp long. It encodes 576 amino acids with a relative molecular weight of 61.27 KD and a theoretical isoelectric point of 5.51 'untranslated region (UTRR) of 101 BP. The amino acid sequence contains the typical characteristic sequence of HSP60 family. The results of real-time fluorescence quantitative PCR show that the amino acid sequence contains the characteristic sequence of HSP60 family. The induced expression of Ld-HSP60 in female and male adults of potato beetles was not detected under low temperature stress (-10 鈩，

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