《环境昆虫学报》

作者	单位
郑锦龙，许小霞，余静，张展滔，兰可可，仇培，金丰良	华南农业大学农学院，广东省生物农药创制与应用重点实验室，广州 510642

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中文摘要:

整合素(Integrin)是一种由α和β亚基组成的跨膜异二聚体蛋白，在昆虫血细胞对外物的包囊过程中起着重要的作用。为阐明integrinβ1参与小菜蛾Plutella xylostella体内细胞免疫中的功能，本研究利用RT-PCR结合RACE技术克隆了小菜蛾integrinβ1基因的cDNA全长序列，命名为PxIntβ1(GenBank登录号GQ178290)。小菜蛾PxIntβ1的cDNA全长序列为2 832 bp，开放阅读框为2 487 bp，编码828个氨基酸，成熟的氨基酸序列中有一个跨膜区和一个integrin亚基。系统进化树显示小菜蛾PxIntβ1与亚洲玉米螟Ostrinia furnacalis整合素的同源性最高，两者同源性达到78%。利用半定量RT-PCR技术检测PxIntβ1基因在小菜蛾不同发育历期(卵、1-4龄幼虫、蛹、成虫)、不同组织(血细胞、体壁、脂肪体、中肠、马氏管)和细菌(金黄色葡萄球菌和大肠杆菌混合菌液)诱导下的表达模式。半定量结果表明小菜蛾PxIntβ1基因在小菜蛾整个发育历期除了卵之外都有表达，4龄幼虫转录水平最高，在血细胞中特异性的高表达，混合细菌诱导2-48 h后，PxIntβ1基因在小菜蛾诱导后24 h达到诱导表达高峰。为了进一步获得重组蛋白PxIntβ1，本研究构建了原核表达质粒pET-32a-PxIntβ1，融合蛋白在大肠杆菌Eschericha coli BL21中获得高效表达，Ni-NTA一步纯化了融合蛋白，并免疫新西兰大白兔，获得了多克隆抗体anti-PxIntβ1。SDS-PAGE电泳和Western blot分析结果表明，His抗体和多克隆抗体anti-PxIntβ1能特异性识别融合蛋白PxIntβ1；进一步利用显微镜观察了小菜蛾血细胞对凝胶珠Sephadex-A25的包囊情况，结果表明抗血清anti-PxIntβ1处理过的小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用受到明显抑制，重组蛋白PxIntβ1可以提高小菜蛾血细胞对Sephadex-A25凝胶珠的包囊作用；以上研究结果表明小菜蛾PxIntβ1在小菜蛾细胞免疫中起着重要的作用。

英文摘要:

Integrin is a transmembrane protein dimers composed by alpha and beta subunits, integrin has been reported to play a vital role in hemocyte encapsulation foreign objects in insects. In order to illustrate the function of integrinβ1 involved in cellular immunity of P. xylostella. In the present study,a full-length cDNA of an integrinβ1 gene was cloned from P. xylostella by RT-PCR and rapid amplification of cDNA ends (RACE) techniques,and designated as PxIntβ1(GenBank accession no.: GQ178290). Its full-length cDNA is 2 832 bp, and the open reading frame (ORF) is 2 487 bp, encoding 828 amino acid residues. The mature amino acid sequence of PxIntβ1 is composed of a transmembrane region and an integrin subunit. Phylogenetic analysis indicated that PxIntβ1 has the highest homology to integrinβ from O. furnacalis, the similarity between them is 78%. Semi RT-PCR was employed to analyze the expression profiles of this gene in different tissues (hemocyte, epidermis, fat body, midgut and malpighiam tubules) of the 4th instar larvae, different developmental stages（egg, 1st-4th instar larva, pupa, and adult）and microbial induction (the mix between Staphylococcus aureus and Escherichia coli）in P. xylostella. Semi RT-PCR results indicated that the mRNA level of PxIntβ1 was expressed during the whole developmental stages except eggs, with the highest transcript level in the 4thinstar larva. Tissue specific result showed that PxIntβ1 was specifically expressed in hemocyte. Microbial induction result showed PxIntβ1 was induced significantly by mixed bacteria from 2-48 h,with the highest expression level at 24 h post-injection mixed bacteria.In order to acquire the recombinant protein of PxIntβ1E.coli BL21. and recombinant protein was one step purified by Ni-NTA.The polyclonal antibody anti-PxIntβ1 with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit. SDS-PAGE and western blot analysis investigated that fusion protein PxIntβ1 can be specifically recognized by both His-antibody and polyclonal antibody anti-PxIntβ1. The encapsulation assay of the hemocyte of P. xylostellaagainst Sephadex-A25 gel beads was observed by microscope. The results investigated that the encapsulation of P. xylostella hemocyte against Sephadex-A25 gel beads was significantly repressed after polyclonal antibody anti-PxIntβ1 incubation with hemocyte, but the encapsulation of P. xylostellahemocyte against Sephadex-A25 gel beads was significantly enhanced after recombinant protein PxIntβ1 incubation with hemocyte. All the above results indicated that PxIntβ1 plays an important role in cellular immunity of P. xylostella.

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