Study On The Construction Of Expressing Vector With Ipt Gene And Its Genetic Transformation In Rice

Leaf senescence is the final stage of leaf development. This process is also of great practical value because during leaf senescence, nutrients are recycled to other parts of the plant. However, in an agricultural setting, leaf senescence may limit yield in certain crops.During the late growth period of rice, premature leaf senescence decreases photosynthetic rate, which lowers the assimilation capability gradually and prevents rice yield from further increase. Cytokinin is an important phytohormone involved in delaying senescence of plant leaf, and isopentenyl transferase (ipt) gene encodes the key enzyme that catalyzes cytokinin biosynthesis. Transformation of chimeric gene which consists of promoter specific to leaf senescence expression and ipt gene can specifically increase cytokinin of transgenic rice, delay leaf senescence effectively and find a new route that can increase the yield of rice.In this study, the ipt gene and PSAG12 promoter which were cloned, were constructed to plasmid vector. And then the recombinant plasmid was transformed into embryos of rice cultivar via Agrobacterium tumefaciens system and relative molecular tests were conducted.The main results of this research are as follows:1. The ipt gene from the plasmid in Agrobacterium tumefaciens (strain C58) was successfully isolated, cloned and sequenced, which contained the whole open reading frame with 723 base pairs. Compared with the reported ipt gene (AE009419), the homology was 100%.2. 5'flanking sequence of SAG12 gene in Arabidopsis was isolated by polymerase chain reaction. It is composed of 1522 base pairs. Compared with the reported promoter of SAG12 gene (ATU37336), the homology was 99%.3. Target gene ipt and the promoter PSAG12 were acquired through digestion, electrophoresis and recover. And then, the acquired ipt gene and PSAG12 promoter were inserted into the corresponding position of the expression vector pCAMBIA1301. The recombinant plasmid (pCAMBIA1301-SAG12-ipt) was regulated by promoter SAG12 and ipt gene.4. The plant expression was into Agrobacterium tumefaciens strain EHA105 and transformed into embryos of rice cultivar Zhonghua 16 and Zheda H02-117 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing hygromycin and 37 resistant plants were acquired. PCR amplification demonstrated that the target gene was integrated into 4 resistant plants.