小菜蛾表皮蛋白基因克隆及表达分析

发布时间：2019-02-21 18:57

【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c DNA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 m RNA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 k D,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]:[objective] to clone the epidermal protein gene (Plutella xylostella cuticular protein,PxyICP) of Plutella xylostella and analyze its sequence characteristics and developmental expression pattern. In order to study the physiological function of PxyICP in the epidermis formation of diamondback moth (Plutella xylostella), the PxyICP, was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of c DNA terminal (RACE). The sequence characteristics were analyzed by bioinformatics, and the relative expression of PxyICP m RNA in diamondback moth (Plutella xylostella) at different developmental stages was studied by real-time fluorescence quantitative PCR. [results] the length of open reading frame of PxyICP gene cloned was 531 bp,. It encodes 177 amino acids with relative molecular weight of 18.90 KD and isoelectric point of 5.32. The results of amino acid sequence analysis showed that the amino acid at position 1 ~ (16) of Plutella xylostella epidermis protein was a signal peptide involved in transmembrane protein transfer, and it had the typical characteristics of conserved motif of RR-1 type in CPR family of insect epidermal protein. The results of real-time fluorescence quantitative PCR analysis at different developmental stages showed that the expression of PxyICP was different in different developmental stages of Plutella xylostella, in which PxyICP was found in the 3rd and 4th instar larvae of Plutella xylostella. The expression levels in pupae and adult were 3.19 and 12.32 times of that of the first instar larva, respectively. [conclusion] the PxyICP gene was cloned successfully. According to its developmental expression pattern, PxyICP protein is involved in the physiological process of epidermis sclerosis and melanization of Plutella xylostella (Plutella xylostella).
【作者单位】：仲恺农业工程学院农业与生物学院;
【基金】：国家自然科学基金青年科学基金项目(31401805) 广东省高等学校优秀青年教师培养项目(KA1548850)
【分类号】：S433.4

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