【作者】 樊东；

【导师】 赵奎军；

【作者基本信息】 东北农业大学 ， 作物遗传育种， 2004， 博士

【摘要】 昆虫几丁质酶cDNA序列可以转化植物或者插入到病原微生物(如Bt或杆状病毒)中并使之表达,增加杀虫效果。对昆虫几丁质酶基因cDNA序列的克隆、表达等一系列分子生物学研究将为生产这类生物杀虫剂奠定重要的理论基础,为害虫的生物防治开辟新的途径。 甜菜夜蛾、粘虫、玉米螟、二化螟四种昆虫均是重要的农业害虫,每年给农业生产都造成一定程度的损失,对这些害虫的生物防治宜采用以苏云金芽孢杆菌为主的多种生物杀虫剂的综合使用的策略。由于昆虫几丁质酶所具有的潜在杀虫活性,可以利用上述昆虫的几丁质酶与苏云金芽孢杆菌的协同杀虫作用来有针对性地增强对甜菜夜蛾、粘虫、玉米螟和二化螟的杀虫效果,并且希望通过此途径有效地避免这些害虫抗性的产生。 本实验从预蛹期甜菜夜蛾、粘虫、玉米螟、二化螟四种昆虫体内各分离克隆了一条几丁质酶基因序列,序列长度分别为2838、2810、3343、2237个碱基,分别包括一个1674、1677、1662和1659个碱基的的开放读码框,编码557、558、553、552个氨基酸,分子量分别为62.6、62.8、61.8和61.4kDa,等电点分别为5.2,5.05,5.28和5.17。 由四种害虫几丁质酶核苷酸序列推导的氨基酸序列为多结构域序列,包括N-端的活性催化区(Catalytic domain),C-端富含半胱氨酸的几丁质结合区(Chitin-binding domain 2)。序列比对表明,在催化区中含有两个高度保守的区域,一个是KFMVAVGGWAEGS,另一个是YDFDGLDLDWEYP,而第二个高度保守区是酶的催化活性中心区。在催化区和几丁质结合区中间有一个被称为Linker的富含脯氨酸、丝氨酸、苏氨酸和谷氨酸的区域(PEST rich domain)。除了几丁质酶催化区、几丁质结合区和Linker区外,在克隆的昆虫几丁质酶N-端成熟蛋白的前端还都含有一个疏水性、大约在20个氨基酸左右的信号肽。 昆虫几丁质酶是一种糖蛋白,糖基化对酶的活化是至关重要的。Linker区一般要经过O-位的高度糖基化修饰,而在催化区也会有部分N-位糖基化修饰。利用NetOGlyc 2.0软件发现在甜菜夜蛾几丁质酶氨基酸序列中有23个O-位糖基化位点,粘虫几丁质酶氨基酸序列中有21个O-位糖基化位点,而玉米螟和二化螟几丁质酶氨基酸序列中均具有29个O-位糖基化位点。N-位糖基化修饰的位点数目也是不一样的,甜菜夜蛾、粘虫和玉米螟3种昆虫几丁质酶氨基酸序列中存在2个N-位糖基化位点,二化螟几丁质酶氨基酸序列中存在3个N-位糖基化位点。除O-位和N-位糖基化位点以外,昆虫几丁质酶氨基酸序列中还存在多个硫酸化和磷酸化位点,这些位点可以通过Expasy分子生物学服务器上的Prosite软件找到,它们的存在对于蛋白功能的发挥均起到一定的作用。 通过NCBI上的Blast软件比较分析,甜菜夜蛾几丁质酶氨基酸序列与烟草天蛾几丁质酶氨基酸序列同源性达到81%,与棉铃虫几丁质酶氨基酸序列同源性达到87%,与斜纹夜蛾几丁质酶氨基酸序列同源性达到96%,与美国白蛾几丁质酶氨基酸序列同源性达到83%,与其他鳞翅目昆虫几丁质酶氨基酸序列同源性达到也达到75%以上。其他3种昆虫几丁质酶氨基酸序列与已经发表的昆虫几丁质酶氨基酸序列进行比较也得到了类似结果,说更多还原

【Abstract】 Several insect chitinase specific cDNAs have been isolated and integrated into transgenic plants to enhance their defence mechanisms, and inserted into insect pathogens to increase their deleterious effects on insects. We reported here the cloning and expression of four insect chitinase cDNAs from the beet armyworm, Spodoptera exigua (Hübner), Oriential armyworm, Mythimna sepatata( Walker), Asian corn borer, Ostrinia furnacalis (Guenee) and rice stem borer, Chilo suppressalis (Walker). A detailed understanding of these chitinase molecular characteristics was necessary for the development of insect chitinase-based insecticides.Four chitinase cDNAs were isolated from the prepupae of S. exigua, M. sepatata, O. furnacalis and C. suppressalis, respectively. The S. exigua chitinase cDNA, 2838 base pairs in length, contained an open reading frame of 1674 base pairs and it encoded a polypeptide of 557 amino acid residues with a predicted molecular weight of 62.6 kDa. The M. sepatata chitinase cDNA, 2810 base pairs in length, contained an open reading frame of 1677 base pairs and it encoded a polypeptide of 558 amino acid residues with a predicted molecular weight of 62.8 kDa. The O. furnacalis chitinase cDNA, 3343 base pairs in length, contained an open reading frame of 1662 base pairs and it encoded a polypeptide of 553 amino acid residues with a predicted molecular weight of 61.8 kDa. The C. suppressalis chitinase cDNA, 2237 base pairs in length, contained an open reading frame of 1659 base pairs and it encoded a polypeptide of 552 amino acid residues with a predicted molecular weight of 61.4 kDa. Two conserved domains were revealed in anyone of the deduced amino acid sequences. One was the highly conserved catalytic domain at the N-terminus, and the other was chitin-binding type 2 domain at the C-terminus which contained six conserved cysteines. There was a Linker named Pro/Glu/Ser/Thr-rich (PEST) region between catalytic domain and chitin-binding domain 2. In anyone of these four insect chitinase amino acid sequences, a hydrophobic signal peptide was predicted to precede the N-terminal region of the mature protein.Several putative N-linked glycosylation and O-glycosylation sites that might be necessary for the secretion of the protein and maintenance of their stability were found within the deduced amino acid sequences of the isolated insect chitinases. Two putative N-glycosylation sites were found in the amino acid sequence of S. exigua1 M. sepatata and O. furnacalis while three putative W-glycosylation sites were found in the amino acid sequence of C. suppressalis, respectively. Moreover, the linker domains of these four insect chitinase amino acid sequences were extensively modified by O-glycosylation. There were 23, 21, 29, 29 O-glycosylation sites in the chitinase amino acid sequences from S. exigua, M. sepatata, O. furnacalis and C. suppressalis by NetOGlyc V 2.0 software, respectively.A number of insect chitinases with high sequence homology were gained by BLAST search. The deduced amino acid sequence of chitinase from S. exigua shared 81% identical amino acid residues in the mature part with Manduca sexta, 87 % with Helicoverpa armigera, 83% with Hyphantria cunea and 96% with Spodoptera litura. The comparison of these four deduced amino acid sequences with other lepidopteran insect chitinases also showed high degree of similarity (over 75%). The comparison suggested that the cDNAs encoded chitinase proteins of S. exigua, Mythimna sepatata, Ostrinia fumacalis and Chilo suppressalis The features indicated that these chitinases belonged to family 18 glycosyl hydrolase. These novel chitinase cDNAs, designated as Sechi, Mechi, Ofchi and Cschi, were submitted in GenBank under with accession No. AY658731, No. AY508698, No. AY726548 and No. AY705930.The recombinant protein from the cDNAs of S. exigua and M. sepatata were obtained using two E. co//-based expression systems and one Pachia pastoris yeast expression system. The chitinolytic activities of the recombinant proteins expressed in E. coli and yeast were analyzed by a colorimetric assay method and the result indicated that the recombinant proteins showed apparant levels of chitinolytic activities using CM-Chitin-RBV as the assay substrate.Transcript analysis of S. exigua chitinase cDNA during various developmental stages and in different tissues was determined by semi-quantitative RT-PCR. A specific product was obtained at all stages, but the relative intensity of the band varied at each stage, indicating different mRNA levels. During the larval-larval, larval-pupal transformation, a clear expression of 5. exigua chitinase mRNA could be observed. However, a detectable level of chitinase transcript was also present at the stages when the larvae were not moulting. The tissue specific expression of chitinase transcript was studied in the integument, midgut and the fat bodies by semi-quantitative RT-PCR in the first and second day of sixth instar larvae. The relative gene expression levels from the first day of the sixth instar larva in fat bodies, midgut and integument were not different while in the second day of the sixth instar larva the relative gene expression levels varied significantly. The relative mRNA expression levels in the gut were 2.3 and 1.3 fold to that from fat bodies and integument.更多还原