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【目的】橘小实蝇Bactrocera dorsalis是世界性分布的危害果蔬的重要检疫性农业害虫,目前已对包括新烟碱类在内的多种杀虫剂产生了抗性。本研究在克隆鉴定橘小实蝇烟碱型乙酰胆碱受体(n AChR)α9亚基基因c DNA的基础上,对其分子特性和系统发育进行生物信息学分析,并检测了该基因在橘小实蝇不同发育阶段及成虫不同组织中的表达模式,为进一步研究其潜在功能及在抗药性中的作用奠定基础。【方法】通过高通量测序技术对橘小实蝇进行转录组测序,对高质量序列拼接组装、基因鉴定及同源性比对分析,预测橘小实蝇烟碱型乙酰胆碱受体候选基因。采用RTPCR和RACE(rapid-amplification of c DNA ends)技术克隆该基因的c DNA全长序列,利用生物信息学分析软件分析其基本生物信息;以α-tubulin为内参基因,利用q PCR研究该基因mRNA在橘小实蝇不同发育阶段及成虫头、胸、腹等组织中的表达模式。【结果】根据预测的基因序列,设计特异性引物进行RACE扩增,从橘小实蝇中克隆获得一条烟碱型乙酰胆碱受体基因的全长序列,c DNA全长1 486 bp,完整开放阅读框1 281 bp,编码426个氨基酸,推测其蛋白质分子量为49.1 k D,理论等电点6.56。该基因经序列比对命名为Bdα9,Gen Bank登录号为JQ178254。氨基酸同源性及系统进化树分析显示,该基因的编码蛋白具有n AChRα亚基的典型特征,并与Agα9和Dmβ3聚类在一起,与其他昆虫n AChRα9亚基具有22%~27%的氨基酸序列一致性。q PCR结果表明,Bdα9mRNA在橘小实蝇整个发育阶段均有表达,成虫期的表达量显著高于卵、2龄幼虫、3龄幼虫和蛹期;Bdα9在橘小实蝇成虫头部中表达量最高,且显著高于胸部和腹部中的表达量。【结论】鉴定了橘小实蝇烟碱型乙酰胆碱受体基因Bdα9,明确了该基因在橘小实蝇不同发育阶段及成虫不同组织中的表达模式。根据q PCR的结果,推测Bdα9可能在橘小实蝇成虫期具有重要功能。
【OBJECTIVE】 Bactrocera dorsalis is an important quarantine agricultural pest which is harmful to fruits and vegetables worldwide. At present, it has become resistant to many kinds of insecticides including neonicotinoids. In this study, based on the cloning and identification of c DNA of nAChR α9 subunit gene, the molecular characterization and phylogenetic analysis of the gene were conducted. The expression patterns of fruit flies in different developmental stages and different adult tissues provide the basis for further study of their potential functions and their role in drug resistance. 【Method】 The transcriptome of Trichosanthes niloticus was sequenced by high-throughput sequencing. The high-quality sequence assembly, gene identification and homology comparison were used to predict nicotinic acetylcholine receptor candidate genes in the fruit fly. The full-length cDNA sequence of c DNA was cloned by RTPCR and rapid amplification of cDNA ends (RACE). The bioinformatics analysis software was used to analyze the basic bioinformatics. Using α-tubulin as an internal control gene, mRNA expression patterns in different developmental stages and adults head, chest, abdomen and other tissues of the orange fruit fly. 【Result】 According to the predicted gene sequence, a specific primer was designed for RACE amplification. The full-length cDNA sequence of nicotinic acetylcholine receptor gene was cloned from Trichosanthes flies. The cDNA was 1 486 bp in length and fully open reading Box 281 bp, encoding 426 amino acids, suggesting that the protein molecular weight of 49.1 kD, the theoretical isoelectric point of 6.56. The gene was named as Bdα9 by sequence alignment and Gen Bank accession number was JQ178254. Amino acid homology and phylogenetic tree analysis showed that the encoded protein of this gene has typical features of nAChRα subunit and is clustered with Agα9 and Dmβ3 and has 22% -27% amino acids with other insect nAChRα9 subunits Sequence identity. q PCR results showed that Bdα9 mRNA was expressed throughout the developmental stages of the fruit fly, the expression level of Bdα9 was significantly higher than that of eggs, 2nd instar larvae, 3rd instar larvae and pupae; Bdα9 was expressed in the head of adult Trichophaea fortunei The highest amount, and significantly higher than the expression in the chest and abdomen. 【Conclusion】 The Bdα9 gene of nicotinic acetylcholine receptor was identified in the orange pest flies, and its expression pattern in different developmental stages of adult Trichodina and its adult tissues was confirmed. According to qPCR results, it is speculated that Bdα9 may play an important role in the adult stage of the orange fruit fly.