小菜蛾表皮蛋白基因克隆及表达分析

Cloning and expression of Plutella xylostella(Linnaeus) cuticular protein gene

  • 摘要: 目的克隆小菜蛾表皮蛋白基因(Plutella xylostella cuticular protein,PxyICP),分析其序列特征和发育表达模式,为深入研究PxyICP在小菜蛾表皮形成中的生理功能提供科学参考.方法以小菜蛾为试验材料,采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆PxyICP,以生物信息学方法对其序列特征进行分析,并采用实时荧光定量PCR研究PxyICP mRNA在小菜蛾不同发育时期中的相对表达量.结果克隆获得的PxyICP基因开放阅读框长531 bp,编码177个氨基酸,相对分子质量约18.90 kD,等电点为5.32.氨基酸序列分析结果表明,小菜蛾表皮蛋白第1~16位氨基酸是参与跨膜蛋白转移的信号肽,且该序列具有昆虫表皮蛋白CPR家族中RR-1型保守基序的典型特征.不同发育时期的实时荧光定量PCR分析结果表明,PxyICP在小菜蛾各发育时期的表达量不同,其中PxyICP在2、3和4龄幼虫、蛹和成虫中的表达量分别是1龄幼虫的2.43、0.51、0.63、3.19和12.32倍,以在成虫的表达量最高.结论成功克隆获得PxyICP基因,根据其发育表达模式推测PxyICP蛋白参与小菜蛾成虫表皮的硬化和黑化等生理过程.

     

    Abstract: ObjectivePlutella xylostella(Linnaeus)cuticular protein gene(PxyICP)was cloned,its sequence cha-racters and developmental expression pattern were analyzed to provides reference for the physiological function of PxyICP in the formation of cuticular in P.xylostella.MethodP.xylostella was used as materials,PxyICP was cloned by reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification cDNA ends(RACE).PxyICP sequence charac-ters were analyzed by bioinformatics method.The relative expression levels of PxyICP mRNA were investigated by real-time fluorescent quantitative RT-PCR method at different develomental stages.ResultThe cloned PxyICP contained 531 bp open reading frame in length,encoded 177 amino acids. Its relative molecular mass was 18.90 kDa,and isoelectric point was 5.32.Amino acid sequence analysis showed that the 1stto the 16thamino acids of cuticular protein was a signal peptide that participated in transmembrane protein transfer.This sequence presented typical characteristics of RR-1 conser-vative base sequence in the CPR family of insect cuticular protein.According to real-time fluorescent quantitative PCR analysis at various development stages,expression of PxyICP differed at each developmental stage.In 2ndinstar larva,3rd instar larva,4thinstar larva,pupal and adult,expression levels of PxyICP were 2.43,0.51,0.63,3.19 and 12.32 times as that in 1stinstar larva.And the expression level reached the peak in adult.ConclusionGene PxyICP is cloned successfully. According to its development expression pattern,protein PxyICP is involved in the cuticle sclerotization and melanogene-sis for P.xylostella adult.

     

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