Stuctural Analysis And Application Research Of PUB19 Promoter

In plant transgenic engineering, the researchers often take means of transforming a exogenous gene into the plant to improve certain quality of plant.The expression levels of foreign gene in plants dependent on the upstream promoterwhich control the its expression feature.Today,the 35S promoter was widely used in transgenic engineering.The exogenous gene can express continously in various tissue of the plants under the control of 35S promoter and the expression levels is stable.However the sustained expression of exogenous gene may lead to accumulation of encoding products,which lead some negative impact on the growth of plant, such as awaste of resources or increasing the burden of plant.Therefore,finding a promoter that can specificly drive a exogenous gene to expression is important for improve the growth of the transgnisc plants to ease the burden of plant metabolism and enhace the quality of plants of The PUB19 promoter which can be induced by low temperature, drought, high salt and ABA. In this study, we constructed a vector pCambia-PUB19::IrrE transformed into Agrobacterium EHA105. And then, the plasmid was transferred into colza(Brassica napus) by Agrobacterium-mediated method. Then,to research expression feature of IrrE controled by PUB 19 promoter and to finger out the salt tolerance of combination seedlings, we carried out some salt assay after resistance screening and PCR positive detection. At present,we have obtained results as follow:(1) PUB19 promoter sequences were analyzed by PLACE and PlantCARE database. The results show that PUB19 promoter contains cis-acting elements -such as TATA box,CAAT box-which are necessary for transcription initation. Then, PUB19 promoter also contains a variety of cis-acting elements which are specificity. These cis-acting elements including:ABRE associated with ABA response,HSE associated with hot, MBS associated with drought response and MYB binding sites,GTl-motif acted in response topathogen and NaCl response, TGACG-motifrelated with auxin and salicylic acid response, DRE and MYBCOREcorrsponding to dehydration/low temperature and salt stress response,etc.(2) we constructed the vector pCambia-PUB19::IrrE which have been transformed into Escherichia coli DH5α and Agrobacterium EHA105. The plasmid pCambia-PUB19::IrrE was transferred into colza(Brassica napus) by the method of Agrobacterium-mediated method. We obtain 43 transgenic plants after resistance screening and PCR detection.(3) We detect the relative expression level of IrrE gene by RT-PCR in transgenic plants leaf under a variety of stress conditions. The results shows that IrrE gene under the control of PUB19 promoter can be induced by PEG,Cold,NaCl,ABA and Hot rather than H2O and SA. This result indicated that PUB 19 promoter can be induced by various abiotic stresses in colza.(4) We detect the relative expression level of IrrE gene by RT-PCR. in transgenic plants leaves under differen concentration of NaCl The results shows that the expression level of IrrE gene in transgenic PUB19::IrrE plants can be detected under 100,200,300 and 400 mmol/L NaCl stress,but not 0 mmol/L NaCl stress.the expression level of IrrE genein transgenic PUB19::IrrE plants is higer than transgenic 35S::IrrE plants.This results indicate that the gene expression driven efficient by PUB19 promoter is higher than that driven by 35S promoter.(5) We detect the salt stress resistance of transgenic plans of PUB19::IrrE gene under differen concentration of NaCl. The results shows that the antioxidant enzyme activity,osmotic regulation ability and structural integrity of cell membrance of transgenic plans of PUB19::IrrE gene are better than that of transgenic plans of 35S::IrrE gene under 200,300 and 400 mmol/L NaCl stress.This results indicated that the efficient expression of IrrE driven by PUB19 promoter enhance the salt tolerance of transgenic plans.