西花蓟马化学感受蛋白的cDNA克隆、时空表达分析及组织定位

摘要/Abstract

摘要： 【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FoccCSP1蛋白抗体，与样品切片中FoccCSP1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 FoccCSP 1（GenBank登录号：KM527949）。该基因cDNA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 kD，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。FoccCSP1与东亚飞蝗 Locusta migratoria LmigCSP（GenBank登录号: CAJ01476.1）的氨基酸序列一致性最高，进化关系最近。 FoccCSP 1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pET-30a /FoccCSP 1, 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 FoccCSP 1的核苷酸、氨基酸序列特征。 FoccCSP 1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。

关键词: 西花蓟马, 化学感受蛋白, 分子克隆, 原核表达, 表达谱, 免疫定位

Abstract: 【Aim】 To study the function of chemosensory proteins (CSPs) in the western flower thrips, Frankliniella occidentalis . 【Methods】 A CSP gene from F.occidentalis was cloned using RT-PCR and RACE-PCR strategies. Nucleotide sequence was analysed using DNAMAN software. Homology was analysed using BLAST. A neighbor-joining tree was constructed using MEGA6. Expression profiles of FoccCSP-1 in different developmental stages and different tissues of adults (antennae, head, legs, thorax and abdomen) were assayed using real-time quantitative PCR (qRT-PCR). Polyclonal antisera were prepared by immunizing New Zealand white rabbits with purified protein, and reacted with this protein in slices and 10 nm colloidal gold-affinipure goat anti-rabbit IgG. The immunolocalization of FoccCSP1 protein in adult tissues was clarified by transmission electron microscope. 【Results】 A new chemosensory protein gene was cloned and identified from F. occidentalis and named FoccCSP-1 (GenBank accession number: KM527949). FoccCSP 1 cDNA contains a 288 bp openreading frame encoding a putative protein of 95 amino acids with the molecular mass of 11.377 kD and the isoelectric point of 4.72. The deduced amino acid sequence contains the typical four-cysteine signature like CSPs from other insects. FoccCSP1 shows the highest amino acid sequence identity and the closest evolutionary relationship with LmigCSP (GenBank accession number: CAJ01476.1) of Locusta migratoria. FoccCSP-1 was predominantly expressed in 1-day-old female adults and the 2nd instar nymphae, and also highly expressed in antennae and legs. The recombinant expressed plasmid pET-30a/ FoccCSP 1 was successfully constructed, and the recombinant protein was expressed in a prokaryotic expression system and purified by affinity chromatography using Ni-NTA-Sefinose Column. The immunocytochemistry results indicated that the lymph of antennae, legs and head was strongly labeled by anti FoccCSP1 antisera. 【Conclusion】 The sequence characteristics of nucleotides and amino acids of FoccCSP 1 were clarified. FoccCSP 1 is broadly expressed in multiple developmental stages and adult tissues, suggesting that FoccCSP1 might play an important role in olfactory reception, chemosensory and development regulation of F.occidentalis.

Key words: Frankliniella occidentalis, chemosensory protein, molecular cloning, prokaryotic expression, expression profile, immunolocalization

张治科, 吴圣勇, 雷仲仁. 西花蓟马化学感受蛋白的cDNA克隆、时空表达分析及组织定位[J]. , 2015, 58(1): 1-14.

ZHANG Zhi-Ke, WU Sheng-Yong, LEI Zhong-Ren. cDNA cloning, expression profiling and immunolocalization of a chemosensory protein in the western flower thrips, Frankliniella occidentalis (Thysanoptera: Thtipidae)[J]. , 2015, 58(1): 1-14.

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西花蓟马化学感受蛋白的cDNA克隆、时空表达分析及组织定位

cDNA cloning, expression profiling and immunolocalization of a chemosensory protein in the western flower thrips, Frankliniella occidentalis (Thysanoptera: Thtipidae)

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