【作者】 张欢欢；

【导师】 张建珍； 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2012， 硕士

【摘要】 几丁质是昆虫表皮及围食膜的重要成分,许多酶参与几丁质的合成。研究昆虫几丁质合成途径关键酶的生物学功能,对于解析昆虫几丁质合成机制具有重要意义。本文选择了飞蝗几丁质合成途径的谷氨酰胺：果糖-6-磷酸转胺酶(glutamine: fructose-6-phosphate amidotransferase, GFAT)和葡萄糖胺-6-磷酸-N-乙酰转移酶(Glucosamine-6-phosphate-N-acetyltransferase, GNA),对这两个酶的序列特征,表达特性和生物学功能等进行了研究。我们从飞蝗EST数据库获得GFAT基因的部分cDNA片段,采用5’-RACE技术获得飞蝗GFAT的5’端序列,通过拼接得到了飞蝗GFAT的cDNA全长。该序列长3202bp,开放阅读框ORF为2292bp,编码764个氨基酸,相对分子量为85.6kD。序列分析表明飞蝗GFAT与其他已知昆虫的GFAT蛋白具有较高的序列一致性,但是飞蝗GFAT蛋白N-端多出74个氨基酸。GFAT的催化位点在真核生物与原核生物中都很保守。磷酸化位点预测表明飞蝗GFAT含有5个PKA磷酸化位点S271、S326、S380、S567和S581,可能与PKA及AMPK的磷酸化调节有关。基于己知物种GFAT全长氨基酸序列构建系统进化树,结果表明昆虫GFAT按物种亲缘关系依次聚类,哺乳动物GFAT1和GFAT2形成两个独立的分支。飞蝗GFAT的mRNA表达分析表明,GFAT在飞蝗表皮中表达量最高,其次是脂肪体和翅芽；飞蝗GFAT在5龄表皮的发育表达图谱表明,GFAT在5龄初期表达量最高,随后逐渐降低。5龄飞蝗GFAT的mRNA被干扰后,93%的若虫在蜕皮过程中出现蜕皮困难而死亡的表型。上述研究表明：飞蝗GFAT参与了飞蝗表皮儿丁质的合成,并可能受磷酸化和UDP-N-乙酰葡萄糖胺的反馈抑制调节。采用生物信息学方法,在飞蝗EST数据库中得到飞蝗GNA的cDNA序列,全长1230bp,开放阅读框ORF为612bp,编码204个氨基酸。飞蝗GNA与其他物种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。基于pET-28a(+)质粒构建飞蝗GNA重组蛋白原核表达载体,在大肠杆菌E. coli BL21(DE3)菌株中成功表达飞蝗GNA重组蛋白,经过Ni-NTA亲和柱纯化得到单一目的条带,飞蝗GNA重组蛋白的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200gM时,Acetyl-CoA的Km=133.60μM；当Acetyl-CoA的浓度为200μM时,D-GlcN6P的Km=42.43μM。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。飞蝗GNA被干扰后未观察到对飞蝗蜕皮及发育产生影响,因此,推测可能存在N-乙酰葡萄糖胺激酶(GlcNAc kinase)能代替GNA生成N-乙酰葡萄糖胺-6-磷酸(GlcNAc6P)用于几丁质的合成,从而形成对GNA缺失补救途径。本文针对飞蝗几丁质合成途径两个关键酶谷氨酰胺：果糖-6-磷酸转胺酶和葡萄糖胺-6-磷酸-N-乙酰转移酶,开展基因的cDNA序列分析、表达特性及功能研究,探讨了两个基因在飞蝗发育过程中的作用,研究结果为飞蝗表皮几丁质合成机制的阐明及害虫有效控制提供了重要的基础数据。更多还原

【Abstract】 Chitin was widely distributed in epidermis and peritrophic matrix of insects, many enzymes were involved in chitin biosynthesis pathway. The importance of chitin in insect development and the absence of chitin in vertebrates had led to a hypothesis of attacking insect chitin biosynthesis pathway as a novel target for developing safe and effective insecticides. However, the roles of these enzymes in insect development have not been well elucidated. In this thesis, glutamine:fructose-6-phosphate amidotransferase (GFAT) and Glucosamine-6-phosphate-N-acetyltransferase (GNA) of Locusta migratoria, two key enzymes in chitin biosynthesis pathway, were selected to study their sequences, mRNA expression pattern and biological function.A partial cDNA sequnence of GFAT was obtained from Locust Database, blast analysis showed this sequence had intact3’terminal sequence and miss5’end sequence.5’-RACE PCR was performed to get full length cDNA sequence of LmGFAT. The obtained complete cDNA sequence of LmGFAT was3202bp, contained ORF2292bp encoded764amino acid residues, predicted molecular weight was85.6kD. LmGFAT showed high indentity with other GFATs by animo acid sequence alignment. Howerve, LmGFAT presented additional74amino acid residues at N-terminal compared with other known insect GFATs. The catalytic cites were very conserved in GFATs from both prokaryotes and eukaryotes. Five potential PKA phosphorylation site S271, S326, S380, S567and S581were predicted, which could be involvoed in PKA or AMPK phosphorylation regulation. Phylogenetic tree indicated that GFATs were clustered based on the evolution relationship, in addition, GFAT1and GFAT2of mammal were separated to two different clusters.Tissue specific expression pattern showed that LmGFAT had the highest expression in integument and higher in fat body and wing pad in second day of the5th-instar nymph. The developmental expression pattern in each day of the5th instar nymphs showed that LmGFAT was highly expressed in first day of the5th instar nymphs, and then declined at the following days of the same stage. After LmGFAT was down regulated by RNA interference,93%insects were dead when molting to adult. In conclusion, LmGFAT should be involved in chitin biosynthesis, and regulated by feedback of UDP-GLcNAc and phosphorylation of PKA and AMPK.The cDNA sequence of LmGNA was searched out from Locust database by using bioinformatics methods. The complete cDNA sequence consists of1230bp nucleotides that contain a612bp ORF coding203amino acid residues. The amino acid sequences of GNAs from different species appeared high identity, the residues responsible for binding substrate GlcN6P are exactly same, which suggested GNAs are highly conserved among different species. Recombinant LmGNA protein was expressed in E. coli BL21(DE3) with pET-28a(+) vector containing LmGNA ORF, and then purified by Ni-NTA affinity resin, the enzyme activity was analyzed by spectrophotometer. The optimal enzyme activity was detected at37-50℃and optimum pH was between8.0-9.5. The Km value for D-GlcN6P was42.43μM when the concentration of Acetyl-CoA was200μM, in contrast, the Km value for Acetyl-CoA was133.60μM when the concentration of D-GlcN6P was200μM. Tissue specific expression pattern indicated that LmGNA was mainly expressed in epicuticle, fat body and muscle at the2nd day of the5th-instar nymph. The developmental expression pattern from the1st day to7th day of the5th-instar nymph stage showed that LmGNA was highly expressed after new molting, and then declined at the following days of the same stage. RNAi results indicated the mRNA of LmGNA was effectively silenced, however, ecdysis and development of Locusts didn’t be affected. These data suggested that N-acetylglucosamine kinase, a rescue pathway could be involved in producing N-acetylglucosamine-6-phosphate (GlcNAc6P) instead of GNA for chitin biosynthesis.In this thesis, cDNA cloning, mRNA expression and biological function of LmGFAT and LmGNA were studied. The results would be helpful to improve the understanding of the complexity of insect biosynthesis and explore the chitin metabolic mechanism, which should provide important data for developing new strategies for pest control.更多还原