Cloning And Functional Analysis Of The Genes Encoding Two Enzymes Key To The Isoprenoid Pathway In The Cotton Aphid Aphis Gossypii

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.