Cloning And Functional Analysis Of A Putative UDP-Glucose Pyrophosphorylase From Populus Euphratica And Populus Pruinosa

UDP-glucose pyrophosphorylase(UGPase) is one of the main enzymes that invovle in plant glucose metabolism. The UGPase mainly invovles in the biosynthesis or degradation of UDP-glucose(UDPG) which is the main precursor for the biosynthesis of cell wall carbohydrate components, such as polysaccharide, cellulose and callose.At first, we chose one UGPase-function-like gene for research from4positively selected candidate genes (ESTs) in P. euphratica(data from our laboratory). Then, we chose two different kinds of poplar species as materials, Populus euphratica Olive and Populus pruinosa Schrenk. Both of them are famous for their strong ability to cope with high salinity. By the technique of plant tissue culture, we obtained the fresh calli of both P. euphratica and P. pruinosa. By extracting the total RNA of both P. euphratica and P. pruinosa and RT-PCR, we obtained the full-length cDNA sequences of the UGPase-function-like genes from P. euphratica and P. pruinosa, named PeUGP and PrUGP in this dissertation.The lastest studies about UGPase have showed that many species contain two or more UGPs copies. For example, Arabidopsis thaliana has three UGPs, named as At3g03250, At5g17310and At3g56040. In order to verify PeUGP and PrUGP are two homologous genes, we extracted total RNA from both P. euphratica and P. pruinosa calli lived in five different places. After RT-PCR and TA cloning, finally, we obtained15PeUGP cDNAs from P. euphratica calli,10PrUGP cDNAs from P. pruinosa calli,3PeUGP cDNAs from P. euphratica leaf,4PrUGP cDNAs from P. pruinosa leaf. After phylogenetic analysis, cluster analysis and homology analysis, we verified that the PeUGP and PrUGP are two UGPase-function-like homologous genes, have7different SNPs. As the three-dimensional crystal structure of AtUGPase-A(PDB code2ICY) encoded by gene At3g03250has been determined. Then the primary protein sequences of PeUGPase and PrUGPase were homology modeled by using the crystal structure of UGPase-A(PDB code2ICY). The two predicted three-dimensional structures revealed that both PeUGPase and PrUGPase were structurally highly homologous with AtUGPase-A and they all contain four main domains:the N-terminal domain, the C-terminal domain, the central catalytic domain and the sugar binding domain. They also contain the nucleotide binding (NB) loop. The nucleotide binding (NB) loop is conserved across all pyrophosphorylases.To further examine the expression changes of PeUGPase and PrUGPase under salt stress, we treated P. euphratica calli and P. pruinosa calli with200mM NaCl by0day(Control),1day(short-term, ST),4days(mid-term, MT). The result revealed that in ST and MT, the content of Na+increased in both P. euphratica calli and P. pruinosa calli, but the content of K+fluctuated. Compared with P. euphratica calli, the content of both Na+and K+kept lower in P. pruinosa, but K+/Na+higher. This showed that P. pruinosa has the stronger ability to cope with high salinity than P. euphratica. Then the result of real-time PCR revealed that the content of both PeUGPase and PrUGPase decreased in ST and MT, but it seemed that PrUGPase decreased more heavily, indicating that UGPase may have been involved in plant salt stress and the content of UGPase seems negatively related to plant ability to cope with salinity.