小菜蛾C型凝集素基因PxCTL5的克隆及其在细菌刺激下的转录水平变化

发布时间：2018-01-24 15:48

本文关键词： 小菜蛾 C型凝集素基因克隆微生物诱导免疫应答　出处：《昆虫学报》2017年08期 　论文类型：期刊论文

【摘要】：【目的】本研究旨在克隆小菜蛾Plutella xylostella体内一种C型凝集素基因,检测其在小菜蛾体内的表达模式,并探讨该蛋白对细菌的凝集作用。【方法】基于对小菜蛾转录组和基因组进行生物信息学分析的基础上,RT-PCR结合RACE技术克隆小菜蛾C型凝集素基因全长cDNA序列。构建原核表达质粒载体,在大肠杆菌BL21中高效表达小菜蛾C型凝集素融合蛋白,利用纯化的融合蛋白免疫新西兰大白兔,获得了高效价的抗血清。利用实时荧光定量PCR(RT-qPCR)检测小菜蛾C型凝集素基因在小菜蛾不同发育时期(卵期、1-4龄幼虫、预蛹期、蛹期和成虫期)和小菜蛾4龄第1天幼虫不同组织(血细胞、表皮、脂肪体、中肠和马氏管)中的表达模式。RT-qPCR和Western blot检测小菜蛾C型凝集素基因在大肠杆菌和苏云金芽孢杆菌刺激下的表达差异。显微镜下观察重组蛋白对这两种细菌的体外凝集现象。【结果】克隆了小菜蛾型C型凝集素基因PxCTL5(GenBank登录号:KY807084),cDNA序列全长1 243 bp,开放阅读框(ORF)序列长672bp,编码223个氨基酸残基。RT-qPCR显示PxCTL5基因在小菜蛾各个龄期均有表达,在成虫期处于高水平表达;主要在表皮中表达,可被苏云金芽孢杆菌和大肠杆菌强烈诱导表达,表达高峰期分别为诱导后18 h和24 h。Western blot检测表明,经苏云金芽孢杆菌和大肠杆菌诱导后,小菜蛾血淋巴中PxCTL5蛋白表达量明显提高。体外凝集实验表明,在钙离子存在下,PxCTL5蛋白对两种细菌都有一定的凝集作用,对苏云金芽孢杆菌的凝集作用较强。【结论】小菜蛾PxCTL5可经细菌诱导表达,PxCTL5对苏云金芽孢杆菌有较强凝集作用。
[Abstract]:[objective] to clone a C-type lectin gene of Plutella xylostella Plutella xylostella and to detect its expression pattern in Plutella xylostella. The agglutination of the protein to bacteria was studied. [methods] based on the bioinformatics analysis of the transcriptome and genome of Plutella xylostella. The full-length cDNA sequence of C-type lectin gene of Plutella xylostella was cloned by RT-PCR and RACE, and the prokaryotic expression plasmid vector was constructed. C-type lectin fusion protein of Plutella xylostella was highly expressed in Escherichia coli BL21. The purified fusion protein was used to immunize New Zealand white rabbits. A high titer antiserum was obtained. The C-type lectin gene of diamondback moth (Plutella xylostella) was detected at different developmental stages (egg stage 1-4 instar larva, prepupa stage) by real-time fluorescence quantitative PCR. Pupa stage and adult stage) and diamondback moth larvae on day 1 of 4th instar different tissues (blood cells, epidermis, fat body). Midgut and Markov tube. RT-qPCR and Western. Blot was used to detect the difference of C type lectin gene expression between Escherichia coli and Bacillus thuringiensis. The in vitro agglutination of recombinant protein to these two bacteria was observed under microscope. Plutella xylostella type C lectin gene PxCTL5 (. GenBank login: KY807084. The length of cDNA was 1 243 BP and the length of ORF was 672 BP. The encoding of 223 amino acid residues. RT-qPCR showed that PxCTL5 gene was expressed in all stages of Plutella xylostella and high level in adult. It was mainly expressed in epidermis and could be strongly induced by Bacillus thuringiensis and Escherichia coli. The peak of expression was 18 h and 24 h after induction, respectively. After induction by Bacillus thuringiensis and Escherichia coli, the expression of PxCTL5 protein in haemolymph of Plutella xylostella increased significantly. PxCTL5 protein has a certain agglutination effect on both bacteria, and a strong agglutination effect on Bacillus thuringiensis. [conclusion] PxCTL5 can be expressed by bacteria. PxCTL5 has a strong agglutination effect on Bacillus thuringiensis.
【作者单位】：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;
【基金】：国家自然科学基金项目(31071734,31371989) 广东省科技计划项目(2014A020208106,2015A020209128) 广州市科技计划项目(201509010023)
【分类号】：S433.4
【正文快照】： 脊椎动物依赖高效的获得性免疫系统适应复杂多变的生活环境,而昆虫主要依靠体液免疫和细胞免疫构成的先天性免疫抵抗生存环境中病原微生物的入侵(Vilmos and Kurucz,1998)。体液免疫主要包括经信号转导途径产生合成抗菌效应因子和酚氧化酶原系统的激活(Hultmark,2003;Fauvarqu

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