| 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1 466 bp (GenBank登录号为KC832921)，包括5′非编码区67 bp、3′非编码区268 bp和开放阅读框1 131 bp，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 7 ku，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。 |
| 关键词: 苹果蠹蛾 β-actin基因 RACE技术 实时定量PCR |
| DOI： |
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| 基金项目:国家公益性行业（农业）科研专项“入侵生物苹果蠹蛾监测与防控技术研究”(200903042) |
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| Molecular cloning,sequence analysis and mRNA expression stability detection of β-actin gene in codling moth Cydia pomonella (L.) |
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YANG Xue-qing,ZHANG Ya-lin
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| Abstract: |
| 【Objective】The purpose of this study was to clone the full-length cDNA of β-actin gene from Cydia pomonella (L.) and determine the possibility of the gene as a reference gene.【Method】The β-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 of β-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 1 466 bp (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),having an open reading frame (ORF) of 1 131 bp and encoding 367 amino acid residues.The deduced relative molecular mass of β-actin was 41.793 7 ku and isoelectric point value was 5.29.The protein contained three characteristic features of an actin protein superfamily and retained 6 kinds of functional sites.The sequence shared more than 99% identity with insect actins retrieved from NCBI database.Quantitative and semi-quantitative PCR assays suggested that there was no significant difference (P>0.05) between developmental stages and insecticides induced codling moth.【Conclusion】The β-actin gene could be used as a reliable reference gene in quantifying mRNA expression in C.pomonella. |
| Key words: Cydia pomonella (L.) β-actin gene RACE technique Real-Time PCR |
