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【目的】研究西花蓟马Frankliniella occidentalis化学感受蛋白(chemosensory proteins,CSPs)在其嗅觉及化学感受系统中的作用。【方法】利用RT-PCR和RACE技术克隆西花蓟马化学感受蛋白基因,用DNAMAN软件进行序列分析,使用BLAST进行同源性比较,采用MEGA6的Neighbor-joining法构建了进化树。通过实时定量PCR(real-time quantitative PCR,qRT-PCR)检测了西花蓟马不同发育期以及成虫不同组织(触角、头、足、胸、腹)中该基因的表达谱。免疫新西兰大白兔制备了Focc CSP1蛋白抗体,与样品切片中Focc CSP1蛋白以及10 nm胶体金颗粒偶联的羊抗兔二抗反应,经透射电镜观察,对该蛋白在西花蓟马成虫组织中进行免疫定位。【结果】克隆并鉴定了一个西花蓟马化学感受蛋白基因,命名为Focc CSP1(Gen Bank登录号:KM527949)。该基因c DNA序列全长597 bp,完整开放阅读框(ORF)288 bp,编码95个氨基酸,成熟蛋白分子量11.377 k D,等电点4.72,具有化学感受蛋白典型的4个保守半胱氨酸位点特征。Focc CSP1与东亚飞蝗Locusta migratoria Lmig CSP(Gen Bank登录号:CAJ01476.1)的氨基酸序列一致性最高,进化关系最近。Focc CSP1在西花蓟马不同发育阶段和成虫不同组织中均有表达,在羽化1 d的雌虫中相对表达量最高,其次是2龄若虫,蛹和成虫后期表达量最低;在触角和足中相对表达量较高。成功构建了重组表达质粒p ET-30a/Focc CSP1,并诱导表达,经Ni柱纯化,获得目的蛋白;免疫定位表明,该蛋白在西花蓟马触角、足、头等部位血淋巴中均大量存在。【结论】明确了西花蓟马化学感受蛋白基因Focc CSP1的核苷酸、氨基酸序列特征。Focc CSP1广泛分布在西花蓟马多个组织及各个发育期,据此推测该基因可能在西花蓟马嗅觉识别、感受机械刺激以及调节生长发育等方面扮演重要角色。
【Objective】 The purpose of this study was to investigate the effects of chemosensory proteins (Frankliniella occidentalis) on the olfactory and chemosensory system of western flower thrips (Frankliniella occidentalis). 【Method】 The gene of chemotherapeutic protein of western flower thrips was cloned by RT-PCR and RACE technique. DNAMAN software was used to analyze the sequence. BLAST was used to compare the homology. The phylogenetic tree was constructed by Neighbor-joining method of MEGA6. Real-time quantitative PCR (qRT-PCR) was used to detect the expression profile of the gene in different developmental stages and adult adult worms (antenna, head, foot, chest and abdomen). Focc CSP1 antibody was immunized in New Zealand white rabbits and reacted with Focc CSP1 protein and 10 nm colloidal gold particles conjugated sheep goat anti rabbit antibody. The results of transmission electron microscope showed that Focc CSP1 protein was expressed in the adult tissues of F. occidentalis Immunolocalization. 【Result】 A FHCL gene was cloned and identified as Focc CSP1 (Gen Bank accession number: KM527949). The cDNA sequence of this gene is 597 bp with a complete ORF of 288 bp encoding 95 amino acids. Site characteristics. Focc CSP1 and Locusta migratoria Lmig CSP (Gen Bank accession number: CAJ01476.1) had the highest identity and evolutionary relationship. Focc CSP1 was expressed in different developmental stages of F. occidentalis and in different tissues of adults, with the highest relative expression in females one day after eclosion, followed by the second instar nymphs and the lowest expression in pupae and adults In the relative expression of higher. The recombinant plasmid p ET-30a / Focc CSP1 was successfully constructed and induced to express, and purified by Ni-column to obtain the target protein. Immunolocalization showed that the protein was abundant in the hemolymph of the western flower thrips . 【Conclusion】 The nucleotide and amino acid sequences of the Focc CSP1 gene of western flower thrips were confirmed. Focc CSP1 is widely distributed in many tissues and developmental stages of F. occidentalis, suggesting that this gene may play an important role in olfactory identification, mechanical stimulation and regulation of growth and development of F. occidentalis.