中文摘要：

【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。

英文摘要：

【Aim】This study aims to clone a C-type lectin gene from Plutella xylostella,to investigate its expression patterns and to elucidate its agglutination on bacteria. 【Methods】Based on the bioinformatical analysis of genome and transcriptome database of P. xylostella,the full-length cDNA of a C-type lectin gene was cloned from P. xylostella by RT-PCR and rapid amplification of cDNA ends（ RACE）techniques. Prokaryotic expression plasmid was constructed and the fusion protein was expressed in E.coli BL21. The polyclonal antibody with high serum titer was prepared using the purified fusion protein to immunize New Zealand white rabbit. Real-time quantitative PCR（ RT-qPCR） was employed to analyzethe expression profiles of this gene in different tissues（ hemocyte,cuticle, fat body, midgut and Malpighiam tubules） of the day-1 4th instar larvae and different developmental stages（ egg,1st-4th instar larva,prepupa,pupa,and adult） of P. xylostella. RT-qPCR and Western blot were used to detect the expression difference of this gene after challenge with E. coli and B. thuringiensis at the mRNA and protein levels, respectively. The agglutination of the recombinant protein to the two bacteria was investigated by microscopic observation. 【Results】A C-type lectin gene was cloned from P. xylostella and named PxCTL5（ Gen Bank accession no. ： KY807084）. Its full-length cDNA is 1 243 bp,with the open reading frame（ ORF） of 672 bp,encoding 223 amino acid residues. RT-qPCR analysis revealed that PxCTL5 was expressed in all developmental stages,with the highest expression level in adults of P.xylostella,and mainly in the cuticle,in which the transcript level was up-regulated upon B. thuringiensis or E. coli challenge,with the highest expression level at 18 h and 24 h post infection,respectively.Western blot results also confirmed that the expression level of PxCTL5 in the hemolymph was significantly increased after challenge with B. thuringiensis and E. coli,respectively. Agglutination assay in vitro sho