Cloning Of Ugt CDNA Of Psammoilene Tunicoides And Bioinformatics

Psammosilene tunicoides, a monotypic genus to Psammosilene tunicoides of Caryophyllaceae, ranks among the most important medical herbs of several famous Chinese patent medicines like Yunnan Baiyao. The effective medical component of Psammosilene tunicoides is the triterpenoid saponins, which has been confirmed to play a key role in immune modulation as well as bacteriostasis and bactericidal actions, and can be used as analgesic and anti-inflammatory. Unfortunately,Psammosilene tunicoides requires strict growth conditions while breeds at a low speed and owns a poor ability of metabolism. On the other hand, it only exists in a small area in south-western China, including Yunnan, Guizhou and Sichuan.Considering of its important pharmacological activities and the danger of exhaustion,Psammosilene tunicoides now has been listed as one of the national second-grade protection species. Undoubtedly, it is an urgent task for scientists to find out new ways for the utilization of Psammosilene tunicoides.To date, with the help of synthetic biology, designing and transforming microbial strains in order to product natural compounds has become a highly effective method for relieving the shortage of medical resources, as the reconstruction of the generating module of triterpenoid saponins of Psammosilene tunicoides in microorganisms helps the heterologous efficient synthesis of the metabolic pathway of the medicinal active components. The key is to realize the above method is to find out the key biological element in cloning the metabolic pathway.Design and Transform Microbial Strains in the biosynthesis process of triterpenoid saponins, UDP-glucuronosyltransferase(UGT) is the key enzyme gene that mediates the modification, as it modifies β-amyrin, the common parent nucleus of triterpenoid saponins, together with the P450 monooxygenase gene, and thus various kinds oftriterpenoid saponins are synthesized. However, as UGT belongs to the supergene family, it is challenging for scientists to do further researches, thus, there are just few relevant reports, none of which are about UGT in Psammosilene tunicoides.Hence, based on the transcriptome data the research group has achieved, here we design an amplification primer of the full-length sequence of UGT gene for cloning and obtain the full-length c DNA sequence, which later is linked with T-vector for sequencing. What’s more, we research on the opening reading frame(ORF) of UGT gene in Psammosilene tunicoides and analyze the physical and chemical properties,the hydrophobic property, the transmembrane domain, the signal peptide, the subcellular localization and the structural domain of the coding protein sequence. Also,we predicate the structure of the protein.Research shows that the full length of c DNA of the cloned UGT in Psammosilene tunicoides is 1581 bp and contains a 1335bp-long opening reading frame(ORF),which encodes 444 amino acids. Predicated by the Bioinformatics, the relative molecular mass and the isoelectric point of the coding protein of UGT in Psammosilene tunicoides is 49.76 KD and 6.25, respectively. The sequence similarity between the UGT gene of Psammosilene tunicoides and of Vaccaria segetalis is 81%.In addition, there exist transmembrane domain and conserved domain of uridine diphosphate glycosyltransferase(UGTs) family in the coding protein sequence of UGT gene in Psammosilene tunicoides, while there are no signal peptides.The Results help characterize the essential physiochemical properties of UGT gene,and reveal the multiplicity as well as the complexity of gene regulation at molecular level. Predictably, further studies on the UGT gene by bioinformatics will provide the basis and conditions for the further functional identification as well as the researches on the expression regulation and the transgene.