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当前位置: 首页 > 文章 > 苹果蠹蛾β-actin基因cDNA的克隆、序列分析及mRNA表达稳定性检测 西北农林科技大学学报(自然科学版) 2014,42 (11) 135-140
Position: Home > Articles > Molecular cloning,sequence analysis and mRNA expression stability detection of β-actin gene in codling moth Cydia pomonella (L.) Journal of Northwest A & F University(Natural Science Edition) 2014,42 (11) 135-140

苹果蠹蛾β-actin基因cDNA的克隆、序列分析及mRNA表达稳定性检测

作  者：
杨雪清;张雅林
单  位：
西北农林科技大学植保资源利用与病虫害防治教育部重点实验室;沈阳农业大学植物保护学院
关键词：
苹果蠹蛾;β-actin基因;RACE技术;实时定量PCR
摘  要：
【目的】克隆苹果蠹蛾(Cydia pomonella(L.))的β-actin基因cDNA全长,确定其作为内参基因的可能性。【方法】运用RT-PCR和RACE技术分段扩增苹果蠹蛾β-actin基因5′和3′非编码区及保守区,拼接后根据所获序列设计引物,完整克隆苹果蠹蛾β-actin基因。利用生物信息学软件对苹果蠹蛾β-actin基因编码的蛋白质进行分析预测;通过实时定量PCR和半定量PCR方法,检测不同发育阶段以及杀虫剂处理后苹果蠹蛾β-actin mRNA的表达情况。【结果】苹果蠹蛾β-actin基因cDNA全长1 466bp(GenBank登录号为KC832921),包括5′非编码区67bp、3′非编码区268bp和开放阅读框1 131bp,编码一个由376个氨基酸残基组成的蛋白质。推导的蛋白质相对分子质量为41.793 7ku,等电点为5.29,含有3个actin蛋白家族的典型识别特征以及6种类型的特定功能位点,氨基酸序列与其他昆虫β-actin一致性高达99%。实时定量PCR和半定量PCR结果表明,β-actin基因在苹果蠹蛾发育不同时期以及杀虫剂处理后表达量无显著差异(P>0.05)。【结论】苹果蠹蛾β-actin基因可作为可靠的内参基因应用于基因mRNA的表达定量研究。
译  名：
Molecular cloning,sequence analysis and mRNA expression stability detection of β-actin gene in codling moth Cydia pomonella (L.)
作  者：
YANG Xue-qing;ZHANG Ya-lin;Key Laboratory of Protection Resources & Pest Management of Ministry of Education,Northwest A&F University;Plant Protection College,Shenyang Agricultural University;
关键词：
Cydia pomonella(L.);;β-actin gene;;RACE technique;;Real-Time PCR
摘  要：
【Objective】The purpose of this study was to clone the full-length cDNA ofβ-actin gene fromCydia pomonella(L.)and determine the possibility of the gene as a reference gene.【Method】Theβ-actin gene was isolated by using RT-PCR and RACE through segmented amplification-splicing-and end to end PCR method,and the sequence was predicted and further analyzed by web-server bioinformatics software.Moreover,to detect the expression ofβ-actin in developmental stages and insecticides-induced codling moth,the Real-Time PCR and semi-quantitative PCR were conducted.【Result】A full-length cDNA fragment with 1 466bp(Accession number:KC832921)of C.pomonella was isolated.The cDNA sequence contained a 67 bp 5′-untranslated region(5′-UTR)and a 268 bp 3′-untranslated region(3′-UTR),havingan open reading frame(ORF)of 1 131 bp and encoding 367 amino acid residues.The deduced relative molecular mass ofβ-actin was 41.793 7ku and isoelectric point value was 5.29.The protein contained three characteristic features of an actin protein superfamily and retained 6kinds of functional sites.The sequence shared more than 99% identity with insect actins retrieved from NCBI database.Quantitative and semiquantitative PCR assays suggested that there was no significant difference(P>0.05)between developmental stages and insecticides-induced codling moth.【Conclusion】Theβ-actin gene could be used as a reliable reference gene in quantifying mRNA expression in C.pomonella.

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