Cloning And Analysis Of Related Genes Involved In The Terpenoid Biosynthesis Pathway Of Tomato

Plant terpenoids play significant roles in plant growth and survival and interactions between plant and entironment.In recent years,the commercial and ecological importance of terpenoids makes their metabolic engineering an attractive subject for investigation,especially,with the great progress in metabolic engineering and regulation mechanism of terpenoids,metabolic engineering of terpenoids based on genetic engineering become to be one of most potential approaches for increasing target terpenoids in plant.However,cloning and identifying of related genes involved in the biosynthesis pathway of terpenoid is very important for genetic manipulation to promote target terpenoids.Tomato is a worldwide vegetable,with the help the terpenoid metabolic engineering, we can improve agronomic traits of tomato,such as quality,flavor,disease and insect resistance.Significant progress of terpenoid metabolic engineering in tomato has been made over the past few years,and some genes from tomato involved in the biosynthesis pathway of terpenoid have been cloned from tomato.However,some key genes still have not been cloned yet.Therefore,this study aims to clone and analysis three genes involved in the terpenoid biosynthesis pathway of tomato,and to enhance the gene expression.The main results were as follows:1.cDNA of isopentenyl diphosphate isomerase 2(IPI2) gene(AB049816) from Nicotiana tabacum as information probe,gain a digital assembly sequence TC183769 from TIGR(The Institute for Genomic Research) tomato database,the cDNA of tomato isopentenyl diphosphate isomerase(SlIPI) was cloned by RT-PCR based on sequence of TC183769,the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number EU253957.Sequencing result show that the full-length SlIPI cDNA contains an ORF length in 708bp, encoding 235aa,show 99%and 100%nucleotide and amino acid sequence identities to TC183769.Homology analysis of tomato IPI showed high homology with IPI from other plants.Predicting analysis online show SlIPI do not have chloroplast transit peptides,its sub-cellular location is cytoplasmic and peroxisomal.SlIPI contains NUDIX domain which is function domain of IPI and have activity site Cys88 and Glu149.3-D structure modeling show that 3-D structure of SlIPI is high similar with human's.Semi-quantitative RT-PCR analysis indicate SlIPI have a high expression levels in different tomato root.Recombinant proteins were expressed in E.coli,its MW is 27.1KD,it is consistent with predicted result by software,overexpressed SlIPI in E.coli can impel terpenoid metabolic flux and promote the biosynthetic ofβ-carotene,thereby,the function of SlIPI have been identified.2.cDNA of 3-hydroxy-3-methylglutaryl coenzyme A synthase(HMGS) gene (NM₁17251) from Arabidopsis thaliana as information probe,gain a digital assembly sequence TC176556 from TIGR tomato database,the cDNA of tomato 3-hydroxy-3-methylglutaryl coenzyme A synthase(SlHMGS) was cloned by RT-PCR based on sequence of TC176556,the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number EU253956.Sequencing result show that the SlHMGS cDNA contains an ORF length in 1389bp,encoding 462 aa,show 99%and 99%nucleotide and amino acid sequence identities to TC172759.Predicting analysis online show SlHMGS contains HMGS activity site. Homology analysis of tomato HMGS showed high homology with HMGS from other plants.3-D structure modeling show that 3-D structure of SlHMGS is high similar with Brassica juncea's.Recombinant proteins were expressed in E.coli,its MW is 51.3KD,it is consistent with predicted result by software.3.cDNA of mevalonate disphosphate decarboxylase(MDC) gene(AF429368) from Hevea brasiliensis as information probe,gain a digital assembly sequence TC172759 from TIGR tomato database,the cDNA of Tomato mevalonate disphosphate decarboxylase(SlMDC) was cloned by RT-PCR based on sequence of TC172759,the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number EU216564.Sequencing result show that the full-length SlIPI cDNA contains a ORF length in 1269bp,encoding 422 aa,show 99%and 99%nucleotide and amino acid sequence identities to TC172759.Homology analysis of tomato MDC showed high homology with MDC from other plants.3-D structure modeling show that 3-D structure of SlMDC is high similar with yeast's.Semi-quantitative RT-PCR analysis indicate SlMDC have a low expression level in whole tomato tissue. Recombinant proteins were expressed in E.coli,its MW is 46.7KD,it is consistent with predicted result by software.