Cloning And Functional Analysia Of UDP Pyrophosphorylase Gene Promoter From Larix Gmelinii

UDP-glucose pyrophosphorylase(UGPase)is an key enzyme in the metabolism pathway of carbohydrate,which catalyzes the reversible reaction of glucose-1-phosphate(Glc-1-P)UTP to unridine diphosphate glucose(UDP-Glc)and pyrophosphate.UDPG is the key precursor for synthesiszing callose and cellulose.Therefore,the synthesis of cellulose can be controlled by regulating the expression of UGPase,which is an effective way to regulate the wood quality.In the previous study,the UDP pyrophosphorylase gene(LgUGPase)was isolated form L.gmelinii,and its functions in enhancing the vegetative growth of plants and increasing the content of cellulose was confirmed through transgenic transformation.For further studying the expression characteristics of LgUGPase,promoter sequence of the LgUGPase gene was isolated form Larix gmelinii and the cis-acting elements were analyzed;On the other hand,plant binary expression vector was constructed,and was introduced into Arabidopsis thaliana via Agrobaterium-mediated genetic transformation.The expression of LgUGPase gene was analyzed by GUS histochemical staining.The mian results are as follows:1.A 1375 bp upstream sequence of LgUGPase was cloned using Tail-PCR.Sequence analysis with the online software PlantCARE showed that the cloned sequence contains TATA-box,CAAT-box and some regulatory elements,as well as light-responsive elements,stress response elements(low temperature and MYB binding sites involved in drought-inducibility),and plant hormones response elements(gibberellin,methyl jasmonate,auxin,etc).The result suggests that the expression of Lg UGPase gene may be regulated by low temperature,gibberellin,methyl-jasmonate and the other factors.2.The pORE-R1-pLgUGPase::GUS binary vector was constructed by homologous recombination method,and was transformed into Arabidopsis thaliana by simiplified in-plant infiltration method.Four stable expression transgenic lines were obtained by genomic PCR and RT-PCR.3.The results of GUS staining of transgenic Arabidopsis thaliana showed that pLgUGPase::Gus was highly expressed in roots,stems and leaves of the transgenic A.thaliana seedlings,and the expression of GUS was significantly decreased in mature plants;which majorly expressed in leaf veins,calyxes,filaments,stamens and fruit pods of mature tissue plants,while no GUS expression was detected in seeds of transgenic A.thaliana.4.Low temperature stress and phytohormone-induced experiments confirmed that the expression of pLgUGPase::GUS was induced by low temperature,methyl jasmonate and gibberellin.The experimental results explained expression characteristics of LgUGPase gene,which provided the theoretical and experimental basis for controlling the wood formation by regulating the expression of the LgUGPase gene.On the other hand,the LgUGPase promoter,an inducible promoter was isolated in this study,which has important application value for genetic improvement of economic crops,especially for grain crops.