Cloning And Fuctional Study Of BnPPT1 Gene In Brassica Napus

Phosphoenolpyruvate(PEP)/phosphate translocator 1 (PPT1) gene is well known for its role as the translocator in importing the important PEP into plastids as substrates for fatty acid biosynthesis and more important for the shikimate pathway. In order to investigate the function of PPT1 from Brassica napus, we genetically isolated a full-length cDNA encoding PPT1 with total RNA of developing seeds from Brassica napus CY-2, and the corresponding genomic DNA region from DNA of young leaves with specific primers designed according to AtPPTl sequences and ESTs from Brassica napus by searching the BBSRC BrassicaDB. This gene, designated BnPPTl, is 2075 bp in length of the genomic DNA with eight introns and is 1224 bp in length of the complete coding sequences encoding 407 amino acids. Multiple sequences alignment of protein sequences showed BnPPT1 shares high homology with AtPPT1 of Arabidopsis thaliana. We constructed the plant expression vector BPE with the full cDNA of BnPPT1 sequence fusioned with the constitutive strong promoter CamV35S and eGFP reporter gene. Using a 401bp fragment of BnPPT1 as the target segment, we built a CamV35S promoter-driven RNAi vector BPTi. Through the method of pollen tube soak, we obtained transgenic Arabidopsis thaliana of these two consturcts to observe the subcellular localization of fusion protein and carried out functional verifications. Subcellular localization analysis showed that proteins encoded by BnPPT1 gene and AtPPT1 protein are both located in the plastids. The BPE transgene completely recovered cuel phenotype caused by the loss of function in AtPPT1 gene. Wihle transgene with RNAi vector BPTi showed an obvious phenotype of low chlorophyll content of leaves due to endogenous AtPPTl inhibited expression. These findings further illustrated that BnPPTl gene and the Arabidopsis AtPPT1 gene are not only highly homologous, but also may have same biological functions. Which thus suggested the gene may play a great role in oil biosythesis in rapeseed. Furthermore, semi-quantitative RT-PCR analysis with RNA isolated from tissues and various stages of developmental seeds demonstrated that BnPPT1 is expressed in oilseeds at middle developmental stages, which indicates that BnPPT1 gene does have an important role in carbohydrate metabolism of developing seeds in Brassica napus. Therefore, by Agrobacterium transfection with oilrape hypocotyls, we made BPE transplants into a cultivar Brassica napus CY2 characterized with high oil content. PCR detection and herbicide resistance screening showed that we have successfully got 32 transgenic oilseeds plants. Oil content and fatty acids composition analysis with self-fertilized mature seeds of TO generation showed that oil contents of the transgenes have a normal similar trend, and most transgenes get higher oil contents in seeds to a maximum increase of 10.5 percentage, while no significant change in fatty acid composition takes place compared with transgenic negative control. However, significant differences of oil content existed among indiviual transplants indicated the location of various transgenes in the genomic may influence its effect. To better understand the transgene function and to provide molecular identities at the same time, with integrated the successes of reports on other species with inverse PCR (IPCR) method and modified several key conditions, the study have successfully isolated and cloned a transgenic Brassica napus exogenous T-DNA flanking sequences of BPE. The results here would greatly facilitate further research and utilization of BnPPT1 gene in improving oil content in Brassica napus.