Cloning And Expression Analysis Of The Uridine Diphosphate Glucose Pyrophosphorylase(UGPase) Gene In Hevea Brasiliensis

Nature rubber is a raw material of industrial and strategic importance.At present,China mainly relies on importing to meet its domestic consumption.Rubber biosynthesis occurs in laticifers and uses sucrose as the sole initial precursor molecule.The regulation of sucrose metabolism in laticifers has a key effect on latex regeneration and rubber production.Uridine diphosphate glucose pyrophosphorylase(UGPase)is one of the main enzymes that involve in plant sucrose metabolism.UGPase mainly catalyzes the production of UDP-glucose from Uridine triphosphate(UTP)and glucose-1-phosphate.UDP-glucose,as the glucose donor,is the key precursor in systhesis of Sucrose and polysaccharides,and pays a key role in growth and development of plant.In this paper,the UGPase gene were cloned for the first time and expressionally characterized in Hevea brasiliensis.The main results were as follows:1.By sequence BLAST in NCBI,two HbUGPases have found in H.brasiliensis and each has two alleles.The full-length cDNA clones of the four HbUGPases were obtained by PCR amplifying,and were named HbUGPase1-1,HbUGPasel-2,HbUGPase2-1 and HbUGPase2-2.2.The four HbUGPase genes encoded proteins of 469 amino acids.The deduced HbUGPases shared amino acid identities of more than 90%,in which that of HbUPase1-1 and HbUPase1-2 was 98.51%and HbUGPase2-1 and HbUGPase2-2 was 99.36%,with only 7 and 3 amino acid differences,respectively.The secondary structure prediction results showed that the HbUGPases all were composed of many alpha helix,random coil and extended strand.The tertiary structure were homology modeled by using the crystal 3D-structure of AtUGPase-A1(PDB ID:21CY)as model.The predicted tertiary structure of the four HbUGPases all contains three main domains:N-terminaldomain,the C-terminal domain and the cental catalytic domain.3.The prokaryotic expression vectors and eukaryotic expression vectors of all four genes were constructed,and genes were expressed with high levels in E.coli and Pichia.4.The expression characters of the HbUGPase genes were studied by real-time quantitative RT-PCR analysis.The HbUGPase1-1 and HbUGPase2-1 genes were expressed in all Hevea tissues,the higher in bark and root.HbUGPase genes expression was up-regulated with Hevea leaf development.During Hevea leaf development,the expression of Hb UGPase2-1 gene was up-regulated more significantly than HbUGPase1-1,suggesting although HbUGPase 1-1 and HbUGPase2-1 function redundantly in regulating leaf growth and development,the HbUGPase2-1 gene plays a major role.The transcripts of HbUGPase1-1 and HbUGPase2-1 in latex are markedly regulated by ethephon and tapping,suggesting the roles in latex regeneration.5.Transgenic Arabidopsis plants of overexpressing the HbUGPase genes were acquired and characterized.The transgenic Arabidopsis plants growed much faster than the wild-type Arabidopsis plants and showed characters of early flowering.In transgenic Arabidopsis plants,the content of sucrose and starch increased significantly compared to the wild-type Arabidopsis plants.Especially for the HbUGP2-1 transgenic lines,the sucrose and starch content increased by 53.2%and 60.93%,respectively,compared with the wild-type Arabidopsis plants.The cellulose content of the four transgenic lines increased significantly,especially for the HbUGPase2-2 transgenic lines that,increased by 36.09%.However the total soluble sugar content did not change much,and the HbUGPase2-1 and HbUGPasel-1 transgenic lines decreased by 6.28%and 26.27%,respectively,and HbUGPasel-2 and HbUGPase2-2 transgenic lines increased slightly.The results of this study lay a foundation for further study of HbUGPase genes and its roles in sugar metabolism and facilitate in the regulation of latex regeneration in H.brasiliensis.