小菜蛾Toll-6基因的克隆及功能分析

为探究Toll-6基因在小菜蛾Plutella xylostella先天免疫中的功能，对克隆鉴定的Toll-6基因序列进行生物信息学分析，利用实时荧光定量PCR检测Toll-6基因的时空表达模式及微生物侵染响应模式，体外验证其在微生物结合、病原相关分子模式（pathogen associated molecular pattern,PAMP）结合和细菌凝集等方面的功能。结果表明：Toll-6基因全长2 313 bp，编码770个氨基酸，预测Toll-6蛋白具有富含亮氨酸重复序列（leucine-rich repeats,LRR）胞外区、跨膜区和Toll/白介素-1受体同源（Toll/interleukin-1 receptor homologous,TIR）胞内等典型Toll受体家族特征；Toll-6基因在不同发育阶段及不同组织中均有表达，其中成虫期表达量最高，4龄幼虫期次之，在4龄幼虫中肠表达量最高；此外，苏云金芽胞杆菌Bacillus thuringiensis菌株Bt8010侵染小菜蛾4龄幼虫6 h后，Toll-6基因表达被显著抑制，而黏质沙雷氏菌Serratia marcesc...

Cloning and functional characterization of Toll-6 gene in the diamond back moth Plutella xylostella

In order to dissect the function of Toll-6 gene in the innate immunity of the diamond back moth Plutella xylostella, bioinformatics analysis of the cloned Toll-6 gene was performed, and the spatio-temporal expression pattern and microbial infection response pattern of Toll-6 gene was detected by quantitative real-time PCR; meanwhile, its functions in microbial binding, pathogen-associated molecular pattern(PAMP) binding and bacterial agglutination were verified in vitro. The results showed that the length of Toll-6 gene was 2 313 bp, encoding 770 amino acid residues, and the amino acid sequence contained leucine-rich repeats(LRR) extracellular region rich in leucine repeats, transmembrane region and Toll/interleukin-1 receptor homologous(TIR) intracellular region characterized by typical Toll receptor family. Toll-6 gene was expressed during different developmental stages, with the highest expression in adults, followed by the 4th-instar larvae, and in different tissues, with the highest expression in the midgut of the 4th-instar larvae. In addition, when the 4th-instar larvae of P. xylostella were infected with Bacillus thuringiensis strain Bt8010 for six hours, Toll-6 gene expression was significantly inhibited. In contrast, when the 4th-instar larvae of P. xylostella were infected with Serratia marcescens strain SmPXG6 for 12 h, Toll-6 gene expression was significantly up-regulated, but suppressed at 18 h after infection. Functional characterization of the protein in vitro demonstrated that Toll-6 protein could bind to bacteria and fungi, as well as peptidoglycan and lipopolysaccharide, although it did not function in bacterial agglutination. In conclusion, Toll-6 as a pattern recognition receptor(PRR) might directly recognize and bind to the PAMP, and initiate the innate immune defense against invasive pathogens in P. xylostella, although the response mechanisms varied with different microbes.