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

【目的】研究飞蝗葡萄糖胺-6-磷酸-N-乙酰转移酶(glucosamine-6-phosphate-N-acetyltransferase,GNA)的特性及生物学功能,为探讨飞蝗表皮几丁质的合成机制及研发新型绿色环保杀虫剂提供实验依据。【方法】采用生物信息学方法,在飞蝗EST数据库中搜索GNA的cDNA序列,并与其它物种GNA序列进行同源比较;设计飞蝗GNA特异性表达引物,分析5龄第2天不同组织样品间的表达差异及5龄第1天至第7天表皮中GNA表达量的变化;构建pET-28a(+)蛋白表达载体,在大肠杆菌E.coli BL21(DE3)菌株中表达GNA重组蛋白,Ni柱亲和纯化重组蛋白并分析重组蛋白的酶学活性;通过RNA干扰研究GNA的生物学功能。【结果】飞蝗GNA与其它物种GNA的氨基酸序列一致性很高,结合底物GlcN6P的氨基酸残基完全一致,说明不同物种间GNA非常保守;飞蝗GNA组织特异性分析显示其在表皮、脂肪体及肌肉中表达量较高;飞蝗5龄表皮的发育变化表明,GNA在刚蜕皮后的表达量最高,之后逐步降低;飞蝗GNA重组蛋白在大肠杆菌E.coli BL21(DE3)菌株中成功表达,经过Ni-NTA柱纯化得到单一目的条带,酶学特性研究表明飞蝗GNA重组蛋白的最适反应温度为37—50℃,最适pH为8.0—9.5;测定了飞蝗重组GNA的Km值,当D-GlcN6P的浓度为200μmol.L-1时,Acetyl-CoA的Km=133.60μmol.L-1;当Acetyl-CoA的浓度为200μmol.L-1时,D-GlcN6P的Km=42.43μmol.L-1;飞蝗GNA的RNA干扰结果显示,GNA的表达能够被有效沉默,但蜕皮及发育未受影响。【结论】飞蝗GNA与其它物种的GNA氨基酸序列一致性很高;用该序列原核表达的重组蛋白具有葡萄糖胺-6-磷酸-N-乙酰转移酶的活性;飞蝗GNA被干扰后未观察到对飞蝗蜕皮及发育产生影响,推测存在N-乙酰葡萄糖胺激酶(GlcNAc kinase)补救途径催化产生GNA的底物N-乙酰葡萄糖胺-6-磷酸(GlcNAc6P),用于几丁质的合成。

【Objective】The objective of this study is to investigate the expression characteristics and biological function of glucosamine-6-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 μmol·L-1 when the concentration of D-GlcN6P was 200 μmol·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.