摘要
表皮蛋白是昆虫表皮的重要组成成份,在昆虫的生长发育过程中起着重要作用,有可能成为农业害虫的防治靶标。双叉犀金龟Trypoxylus dichotomus是铁皮石斛的一种重要害虫,且对其表皮蛋白的研究较少。本文通过研究双叉犀金龟表皮蛋白Td14144的表达纯化及与几丁质结合的相关性质明确Td14144功能的重要性。根据双叉犀金龟转录组测序数据,利用RT-PCR获得表皮蛋白Td14144基因(GenBank登录号:MZ463195),并对其进行生物信息学分析。序列分析表明表皮蛋白Td14144属于CPR家族中RR-2亚族,含有R&R保守结构域;系统进化分析结果表明,Td14144与光肩星天牛Anoplophora glabripennis的AgCP的亲缘关系最近。随后将基因片段与pET-28a载体同源重组构建表达载体pET28a-14144,在大肠杆菌Escherichia coli BL21中原核表达,并使用金属螯合层析进行分离纯化,SDS-PAGE及Western blot验证重组蛋白的表达纯化,成功获得95%以上纯度的Td14144蛋白。利用几丁质结合试验评估Td14144与不同类型几丁质结合的能力,发现Td14144可以与壳聚糖、α-几丁质、β-几丁质和胶体几丁质结合,其中对壳聚糖和α-几丁质的结合能力最强。
Cuticular protein is an important component of insect cuticle.It plays a significant role in the growth and development of insects and may become the target for pest control.Trypoxylus dichotomus is an important pest of Dendrobium officinale,and there are few studies on its cuticular protein.The purpose of this article is to clarify the important functions of cuticular protein Td14144 from T.dichotomus by studying its expression,purification,and chitin-binding ability.Based on the transcriptome of T.dichotomus,the gene of Td14144 was cloned by RT-PCR(GenBank accession number:MZ463195)and subjected to bioinformatic analysis.Sequence analysis showed that Td14144 belongs to the cuticular protein of CPR_RR-2 family with the R&R domain.Phylogenetic analysis showed that Td14144 is closely related to AgCP of Anoplophora glabripennis.Subsequently,the gene was constructed into the pET-28a vector to generate the recombinant expression vector pET28a-14144.The vector was transformed into Escherichia coli BL21 for prokaryotic expression.Td14144 was purified by metal chelation chromatography and analyzed by SDS-PAGE and Western blot.The chitin binding ability of Td14144 toward different types of chitinous substrates including chitosan,α-chitin,β-chitin and colloidal chitin was evaluated.It was found that Td14144 showed the highest affinity to chitosan andα-chitin.
作者
刘晶
包涵
陈磊
刘源涛
刘田
杨青
LIU Jing;BAO Han;CHEN Lei;LIU Yuantao;LIU Tian;YANG Qing(School of Bioengineering,Dalian University of Technology,Dalian 116024,China)
出处
《中国生物防治学报》
CSCD
北大核心
2022年第2期513-520,共8页
Chinese Journal of Biological Control
基金
国家自然科学基金(31830076,31871959)。
关键词
双叉犀金龟
表皮蛋白
几丁质结合蛋白
Trypoxylus dichotomus
cuticular protein
chitin-binding protein
