中国农业科学2012，Vol.45Issue(12)：2393-2403,11.DOI:10.3864/j.issn.0578-1752.2012.12.007
飞蝗葡萄糖胺-6-磷酸-N-乙酰转移酶的特性及生物学功能
The Characteristics and Biological Function of Glucosamine-6-phosphate-N-acetyltransferase in Locusta migratoria
摘要
Abstract
[Objective] The objective of this study is to investigate the expression characteristics and biological function of glucosamine-d-phosphate-N-acetyltransferase (GNA) gene of Locusta migratoria, and to explore the mechanism of chitin synthesis and develop new effective pesticides. [Method] The cDNA sequence of LmGNA was searched from locust EST database by using bioinformatics methods, and multiple alignment was performed for sequence identity analysis. The developmental expression of LmGNA from the 1st to 7th day of the 5th instar nymph stage and the tissue specific expression at the 2nd day of the 5th instar nymph were tested by Real-time PCR. Recombinant LmGNA protein was expressed in transformed E. coli BL21(DE3) with pET-28a(+) vector containing LmGNA ORF, and then purified by Ni-nitrilotriacetic acid (NTA) affinity column and the enzyme activity was analyzed by using spectrophotometer. To better understand the function of LmGNA, RNAi was performed by injection dsRNA of LmGNA into locust nymphs. [Result] The amino acid sequences of GNAs from different species showed a high identity. The residues responsible for binding substrate GlcN6P were exactly the same, which suggested GNAs were highly conserved among different species. Tissue specific expression pattern indicated that LmGNA was mainly expressed in integument, fat body and muscle at the 2nd day of the 5th instar nymphs. The developmental expression pattern from the 1st to 7th day of the 5th instar nymph stage showed that LmGNA was highly expressed after new molting, and then declined at the following days of the same stadium. The recombinant LmGNA protein was successfully heterologous expressed in E. coli as a fusion protein. The optimal enzyme activity was detected at 37-50℃ and pH values 8.0-9.5. The Km value for D-GlcN6P was 42.43 μmol·L-1 when the concentration of Acetyl-CoA was 200 μmol·L-1, in contrast, the Km value for Acetyl-CoA was 133.60 umol·L-1 when the concentration of D-GlcN6P was 200 umol-L'1. RNAi results indicated that the mRNA of LmGNA was silenced effectively, however, ecdysis and development of locust were not affected. [ Conclusion ] GNAs from different species were conserved with high sequence identities. The recombinant LmGNA protein expressed in E, coli presented glucosamine-6-phosphate-N-acetyltransferase activity. The mRNA expression of LmGNA was significantly down-regulated by RNAi, but no visible block of ecdysis or development was observed. These data suggested that N-acetylglucosamine kinase, a reparing pathway could be involved in producing N-acetylglucosamine-6-phosphate (GlcNAc6P) instead of GNA for chitin biosynthesis.关键词
飞蝗/葡萄糖胺-6-磷酸-N-乙酰转移酶/原核表达/蛋白特性/RNA干扰Key words
Locusta migratoria/ glucosamine-6-phosphate-N-acetyltransferase/ prokaryotic expression/ protein characteristic/ RNAi引用本文复制引用
张欢欢,张学尧,刘晓健,马恩波,张建珍..飞蝗葡萄糖胺-6-磷酸-N-乙酰转移酶的特性及生物学功能[J].中国农业科学,2012,45(12):2393-2403,11.基金项目
国家自然科学基金资助项目(30970410)、山西省留学基金项目(2009)、国家公益性行业科技项目(200903021) （30970410）
