The Comparation Of Prokaryotic Expression Between GGPS Genes From Solanum Lycopersicum And S.habrochaites

Lycopene content is one important index of fruit quality, and many researches prove that lycopeneplays an important role in human health, so increasing lycopene content is one aim of tomato breeding.Lycopene is a kind of carotenoids, Enhancing the study of key genes of carotenoid metabolic pathway, ithelps further clear the function of the genes and facilitate tomato quality breeding of improvinglycopene content in fruits.Geranylgeranyl pyrophoaphate sythase gene(GGPS) is an important gene of carotenoidmetabolic pathway in plant. Our previous studies have proved that lycopene content of tomato fruitcould be significantly increased by importing the GGPS gene of S. habrochaites tomato into Solanumlycopersicum. To further analysize the functional differences of the GGPS genes from S. habrochaitesand Solanum lycopersicum, the GGPS genes were separately cloned from S. habrochaites andSolanum lycopersicum, then the prokaryotic expression vectors applied for functionalcomplementation in Escherichia coli were constructed, named as GIB-T and GIB-E respectly. Thedifferences of the two genes were compared by measuring yield of lycopene in the fermentationprocess. So we can get the GGPS gene with high efficiency and find evidences to explain why thelycopene content of tomato fruits between the two materials are different.The main research results are as follows:1. The deduced amino acid sequences of two GGPS genes from S. habrochaites and Solanumlycopersicum were compared. Both of them contained the five conserved domains of geranylgeranylpyrophoaphate sythase, but there were no different amino acids in these five domains between them.While, four amino acids were different in the conserved domain (95aa-363aa) of the isopentenyltransferase enzyme.2. With prokaryotic expression vector pACCRT-EIB (containing three genes of lycopenebiosynthesis pathway，crtE,crtI, crtB), the crtE gene was replaced by GGPS with the same function andtwo prokarytic expression vectors were constructed, named as GIB-T and GIB-E, respectly. The growthcurves of the E. coli DH5α strains with the two vectors were respectly measured and the results showedthat different GGPS genes had no different effect on the growth of bacteria. Relevant genes of lycopenebiosynthesis expressed in E. coli DH5α strain and produced lycopene. The bacteria containing GIB-Twas redder than those containing GIB-E. Aided by High Performance Liquid Chromatography(HPLC), the yields of lycopene were measured every four hours (from12h to48h) in the process offermentation. The contents of lycopene in GIB-T were significantly higher than GIB-E strain.According to the above results, we speculate that the enzyme encoded by GGPS gene from S.habrochaites are more activitve than that from Solanum lycopersicum.3. in order to obtain a new promoter ftom Solanum lycopersicum, the CaMV35s promoter ofplant expression vector pBI121was replaced by Promoter-E, which is the upstream sequence ofGGPS gene from Solanum lycopersicum to construct the transient expression vector pBI121-P-E. The promoter activity of this upstream sequence of GGPS gene was preliminarily determined through thetransient expression by agrobacterium-mediated transformation of tobacco leaf and GUS dyeing.