烟草甲抗菌肽基因Defensin的鉴定及响应细菌的表达模式分析

Identification of Defensin genes and analysis of expression patterns in response against bacteria in Lasioderma serricorne

本研究旨在阐明烟草甲Lasioderma serricorne Defensin(LsDef1和LsDef2)基因在响应细菌侵染过程中的功能。利用生物信息学分析了LsDef1和LsDef2编码蛋白的结构、特征和系统进化关系。采用实时荧光定量PCR(RT-qPCR)技术检测LsDef1和LsDef2在烟草甲不同发育阶段(卵、1龄幼虫、2龄幼虫、3龄幼虫、4龄幼虫、蛹、成虫)、4龄幼虫不同组织(脂肪体、表皮、中肠、血细胞和马氏管)、病原微生物诱导情况、细菌及细菌细胞壁成分胁迫后的表达模式。序列比对发现烟草甲LsDef1和LsDef2具有典型的6个半胱氨酸,形成3对二硫键,属于β防御素。RT-qPCR显示LsDef1和LsDef2基因在烟草甲不同发育阶段均有表达,在幼虫4龄处于高水平表达;主要表达组织为脂肪体(Fat body)和血细胞(Hemocyte),其次为表皮和中肠,而在马氏管的表达水平最低。外来病原物诱导后,LsDef1和LsDef2基因表达量显著增加,对金黄色葡萄球菌Staphylococcus aureus诱导最为敏感,进一步得细菌和细菌细胞壁成分诱导表达模式显示金黄色葡萄球菌诱导高峰期出现在诱导后6 h;PGN-SA诱导表达高峰期为3 h。烟草甲LsDef1和LsDef2是一类可被革兰氏阳性菌诱导的抗菌肽,研究结果为进一步研究昆虫防御素类抗菌肽的功能奠定了基础。

The purpose of this study was to elucidate the function of Lasioderma serricorne Defensin(LsDef1 and LsDef2)genes in response to bacterial infection.The structure,characteristics and phylogenetic relationship of LsDef1 and LsDef2 were analyzed by bioinformatics.Real-time quantitative PCR(RT-qPCR)was used to detect the tissue-specific expression(Fat body,Integument,Midgut,Hemocyte,Malpighian tubule),developmental stages(egg,1st instar larva,2nd instar larva,3rd larva,4th instar larva,pupa,adult)of LsDef1 and LsDef2.RT-qPCR was also to detect the expression pattern of LsDef1 and LsDef2 response against different pathogen,bacteria induction pattern and bacterial cell walls induction pattern.The sequence comparison showed that induction pattern had 6 typical cysteines and formed 3 pairs of disulfide bonds.RT-qPCR showed that LsDef1 and LsDef2 gene in L.serricornes was mainly expression in fat body and hemocytes,followed by integument and midgut,and the expression level was lowest in the Malpighian tubules tissues,and the expression level was high in the 4th instar larvae.It could be significantly stimulated by foreign pathogens,and LsDef1 and LsDef2 were the most sensitive to Staphylococcus aureus.Bacterial and cell wall component induced expression patterns were further analysed,and the expression peak appears at 6 h after induction.When the S.aureus PGN-SA stimulated L.serricorne,the peak expression peak appeared at 3 h after induction.These results indicated that antimicrobial peptides LsDef1 and LsDef2 genes could be induced by pathogen and laid a foundation for further research on the function of LsDef1 and LsDef2.