【作者】 张浩；

【导师】 李正西；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 棉蚜(Aphis gossypii Glover),属半翅目Hemiptera、蚜科Aphididae、蚜属Aphis,是一种世界性的农业害虫,主要通过刺吸植物汁液和传播病毒病对棉花、瓜果蔬菜等造成严重危害。类异戊二烯通路是棉蚜体内一个重要的代谢途径,此通路上的多种类异戊二烯代谢产物对棉蚜的生长发育、生理活动都有重要的作用,因此对此通路上的一些关键酶进行深入的分析和研究具有重要的意义。异戊二烯基焦磷酸合成酶(又称为异戊二烯基转移酶)就是这样一类关键酶,其催化产物是多种类异戊二烯化合物的前体物质。本研究对其中的两个关键酶——十聚异戊二烯焦磷酸合成酶(DPPS)和牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)的编码基因进行了克隆、重组表达和功能分析。十聚异戊二烯焦磷酸合成酶(DPPS)属于长链异戊二烯基焦磷酸合成酶,是辅酶Q异戊二烯侧链合成过程中的关键酶。辅酶Q在生物体中发挥着重要的生理作用。长链异戊二烯基焦磷酸合成酶在细菌、酵母、植物和哺乳动物中已得到广泛研究,但在昆虫中研究得很少。本研究克隆了编码棉蚜十聚异戊二烯焦磷酸合成酶(AgDPPS)两个亚基的基因(AgDPPS1和AgDPPS2, GenBank登录号：KC431243和KC431244)。它们分别包含1230bp和1275bp的开放阅读框,编码409个和424个氨基酸。序列比对和系统进化分析表明,AgDPPS与人类的DPPS类似,是由两个异源亚基构成的。重组表达和体外的酶促分析结果表明,这两个亚基对该酶的催化活性是缺一不可的,形成的主要中间产物是牻牛儿基牻牛儿基焦磷酸(GGPP)。在大肠杆菌DH5α的体内表达分析表明,AgDPPS是棉蚜合成辅酶Q10的关键酶。我们的研究表明,AgDPPS的催化过程包含两个主要步骤,其催化过程以GGPP作为主要中间产物,以十聚异戊二烯焦磷酸(DPP)作为最终产物。我们的研究是昆虫中首次对合成辅酶Q10侧链的关键酶的研究。牻牛儿基牻牛儿基焦磷酸合成酶(GGPPS)是短链异戊烯转移酶,其催化产物牻牛儿基牻牛儿基焦磷酸(GGPP)在蚜虫体内最重要的作用是蛋白质的异戊烯基化。这种修饰对于蛋白质在膜结构或胞质中的特异性定位、细胞骨架组装、细胞间信号传导有重要意义。GGPPS在植物、真细菌、古细菌、酵母和哺乳动物中已被广泛研究,但对昆虫中GGPPS的研究仍然非常少。本研究克隆了编码棉蚜牻牛儿基牻牛儿基焦磷酸合成酶(AgGGPPS)的基因(GenBank登录号KF220654),其具有930bp的开放阅读框,编码309个氨基酸,理论等电点是6.21。序列分析和系统进化分析表明,AgGGPPS的氨基酸序列包含所有异戊二烯基转移酶具有的富含天冬氨酸的保守区域,AgGGPPS作为昆虫的GGPPS被归类为Ⅲ型GGPPS.本研究对AgGGPPS的原核重组表达、亲和层析纯化以及体外酶活性的测定,发现了AgGGPPS能催化DMAPP、GPP或FPP作为烯丙基底物与IPP反应形成GGPP。虽然在不同的底物情况下AgGGPPS的酶活性有所不同,但与其他只接受FPP为烯丙基底物的Ⅲ型GGPPS是不同的。本研究首次鉴定了棉蚜类异戊二烯通路两个关键酶,并对其生化特性进行了深入研究,所获得的结果有助于利用棉蚜类异戊二烯通路调控蚜虫的发育,从而为蚜虫的防控提供科学依据。更多还原

【Abstract】 The cotton aphid, Aphis gossypii Glover (Hemiptera:Aphididae), is an important agricultural pest worldwide. It causes serious harm to cotton, fruits and vegetables by sucking plant juices and spreading viral diseases. Isoprenoid pathway is very important in the metabolism of the cotton aphid. Many metabolites of this pathway have significant effects on cotton aphid’s growth and physiological activities. Therefore, it is necessary to study the key enzymes of this pathway. Specifically, prenyltransferase (polyprenyl diphosphate synthases) plays an important role in the control and regulation of the isoprenoid pathway since its catalytic products are the precursors of many other isoprenoid compounds. In this study, decaprenyl diphosphate synthase (DPPS) and geranylgeranyl diphosphate synthase (GGPPS), two prenyltransferases with great significance to cotton aphid, were studies.DPPS is a long chain polyprenyl diphosphate synthase; all medium-and long-chain polyprenyl diphosphate synthases (PDDSs) catalyze the synthesis of the sidechain prenyl tails of ubiquinones, which play critical physiological roles in all organisms. This class of enzymes has been extensively studied in bacteria, yeast, plants and mammals, but little information is available in insects. Here we cloned the cDNAs encoding the two subunits of an aphid long-chain PDDS (designated as AgDPPSl and AgDPPS2, GenBank accession nos. KC431243and KC431244). AgDPPSl and AgDPPS2had an open reading frame of1230bp and1275bp, with a calculated isoelectric point of8.13and6.28, respectively. Sequence alignment and phylogenetic analysis showed that the enzyme was a candidate decaprenyl diphosphate (DPP) synthase with two heterologous subunits like human DPPS. Recombinant expression and in vitro enzymatic assay revealed that the two subunits were essential for the activity of the enzyme that catalyzed the formation of a major intermediate product geranylgeranyl diphosphate. In vivo analysis of ubiquinone (UQ) by expressing the insect enzyme in Escherichia coli DH5a identified UQ-10. Our data suggested that the insect enzyme is a novel DPP synthase with a two-major step catalytic mechanism, which catalyzes the formation of DPP as the final product, and geranylgeranyl diphosphate as the major intermediate product. This is the first characterization of an insect long-chain DPPS that synthesizes the side-chain of coenzyme Q-10.GGPPS is short-chain prenyltransferase. GGPP is very important to the production of geranylgeranylated proteins. The modification plays a critical role in the specific localization of proteins in the membrane structure or cytoplasm, cytoskeleton, cell signaling and so on. GGPPSs have been extensively studied in plants, eubacteria, archaebacteria, yeast and mammals, but up to now information about an insect GGPPS is still scarce. Here we cloned the cDNA encoding an insect GGPPS from the cotton aphid (designated as AgGGPPS, GenBank accession No. KF220654). AgGGPPS had an open reading frame of930bp, coding for309amino acids, with a theoretical pI of6.21. Sequence and phylogenetic analysis showed that the amino acid sequence of AgGGPPS included the conserved aspartate-rich motifs characterized by all prenyltransferases known to date, and AgGGPPS as a GGPPS of insect could be classified as type-Ⅲ GGPPS. AgGGPPS was over-expressed in E. coli, and recombinant protein was purified by affinity chromatography. In vitro enzymatic activity assay coupled with product identification by gas chromatography-mass spectrometry demonstrated that AgGGPPS could catalyzed the formation of GGPP with DMAPP, GPP or FPP as the allylic cosubstrate in the presence of IPP, suggesting that AgGGPPS accepted not only FPP but also GPP and DMAPP as the allylic cosubstrate. Although the activity of AgGGPPS is different in the case of different allylic cosubstrate, it is different from other type-Ⅲ GGPPSs which accepted only FPP as the allylic cosubstrate.To sum up, two key enzymes in the cotton aphid isoprenoid pathway were identified, and their biochemical characteristics were studied. The data help understand the regulation of the isoprenoid pathway in the cotton aphid, and provide scientific basis for controlling the cotton aphid.更多还原