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为研究赤拟谷盗Tribolium castaneum受到热胁迫后高度保守的热激蛋白70(heat shock protein70,HSP70)基因的表达变化,本研究扩增了681bp的赤拟谷盗hsp70片段,编码227个氨基酸残基,GenBank登录号为HM345948。同源性分析表明:赤拟谷盗hsp70核苷酸序列与马铃薯甲虫Leptinotarsa decemlineata的hsp70(GenBank登录号:AF322911.1)同源性最高,为97%;其推测的蛋白序列与马铃薯甲虫、甘蓝夜蛾Mamestra brassicae、黑腹果蝇Drosophila melanogaster和美洲斑潜蝇Liriomyza sativae的HSP70蛋白均有94%以上的同源性。利用RT-PCR技术得到与赤拟谷盗hsp70进行竞争定量的内部竞争物,以等量的目标cDNA和一系列稀释的竞争模板进行竞争PCR扩增,构建了hsp70的竞争定量PCR检测体系,该体系标准曲线的线性方程为Y=1.032X-1.618(r2=0.975)。这些结果为赤拟谷盗的hsp70定量检测提供了方便,并为热控技术防治害虫提供了基础资料。
In order to study the expression of highly conserved heat shock protein 70 (HSP70) gene in Tribolium castaneum, a 681bp fragment of hsp70 gene encoding 227 amino acids residues GenBank accession number HM345948. Homology analysis showed that the nucleotide sequence of hsp70 of Trichosanthes fulva showed the highest homology with hsp70 (GenBank accession number AF322911.1) of potato beetle Leptinotarsa decemlineata, which was 97%. The deduced protein sequence was homologous to potato beetle, The moths of Mamestra brassicae, Drosophila melanogaster and Liriomyza sativae all had more than 94% homology. The competitive competitor of hsp70 was screened by reverse transcription-polymerase chain reaction (RT-PCR), and competitive PCR was carried out by competitive PCR with an equal amount of target cDNA and a series of diluted competition templates. The linear equation of the system standard curve is Y = 1.032X-1.618 (r2 = 0.975). These results provide a convenient method for the quantitative determination of hsp70 in the red-tarrel and provide the basic information for the thermal control technology to control pests.