Soybean Pep Carboxylase Antisense Gene Cloning And Vector Construction

Soybean is the main source of plant protein and lipid for mankind. With the improvement of standard of living, People are paying more attention to consumption of plant lipid rather than animal ones.How to increase the lipid content of soybean, however is a great challenge for the soybean breeders. Now biotech, which can combine the favorite characters of different species into one variety, provides an effective means to reach the goal. It has mainly three advantages. Firstly, it can not only shorten the cycle of routine of breeding, but also be practicable; secondly, it can transfer the foreign gene into plant acceptor, integrate it into genome of acceptor and eventually express the foreign gene in the plant; and thirdly, it can use gene of different species without restriction of species.Phosphoenolpyruvate carboxylase (PEPCase) is critical enzymes of pyruvic acid metabolism. Studies had confirmed that the prevention of PEP activity can transfer the reaction of substrate into fatty metabolism. This fact presents greatpossiblity to manipulate fatty acid metabolism in plant seeds by gene engineering .Using antisense RNA , which based on nucleic acid hybridization theory, we designs antisense sequence versus special target gene, so the expression of target gene is refrained from expression by the interaction of partnership.In this dissertation, primers are designed according the sequence of PEP registed in Genebank. A DNA fragment of was amplified with polymerase chain reaction (PCR) using soybean totle RNA as template. The gene was sequenced and the results showed that the sequence contained 968 nucleotides and only five different from the PEP gene. We ligase the gene into T-vector and then digist out it at Xmal and EcoRIsites and insert the vector SK(-). In order to make the gene express in plant, the PEP gene fragment was inserted in an antisense orientation using mosaic virus 35S promoter to drive transcription and the nopaline synthase terminator.Crowtoe (Lotus corniculation L.) is the acceptor plant of this antisense gene in this experiment. Plasmid Dantip was mobilized into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method, using that plasmid cloned in E.coli.DH5 α .The antisense PEP gene was transferred into crowtoes via the Agrobacterium mediated method.The antisense PEP was integrated into the acceptor which confirmed by PCR. Among 109 regenerated plants there are 9 contained the transgene.This work has laid a preparation for engineering such gene into soybean in the future. We hope to increase the fatty content of soybean seed by this way finally.