The Plant Expression Vector Construction Of Cotton Chloroplast Cu/Zn-SOD Gene And Genetic Transformation

The problem of earliness with decrepitude has worse effect on the yield in theshort-season cotton in our country. Up to now, about the problem of earliness withdecrepitude in short-season cotton, careful research has been done on the physiologicallevel, the results of the formers: comparing the varieties of earliness with not-decrepitudewith the varieties of earliness with decrepitude, the antioxidant enzyme activity ofearliness with not-decrepitude are higher and steadier in the growth retrial period, the keyenzymes in system of antioxidant enzyme are SOD. If we want to understand themolecular mechanism about earliness with decrepitude, it is very important of cloningthe correlative genes and studying its function. These genes can help us to improve theyield and quality.(1) plant binary vector construction of cotton chloroplast Cu-ZnSOD gene: PlasmidpBI 121 was used as the genetic transformation carrier. The cotton chloroplastCu-ZnSOD genes and pBI 121 was digested by BamHI/SacI, then extracted the genefragments and vector fragment by electrophoresis, linked up the two fragments andtransformed it into Agrobacterium tumefaciens LBA4404 competent cells, which is plantexpression vector, pBI-YSOD. The result of extraction plasmid of pBI-YSOD and PCRshowed that fragment of plant expression vector the same to the target gene, plasmidpBI-YSOD is bigger than plasmid pBI121, which showed that cotton chloroplastCu-ZnSOD gene fragments has be transferred into the Agrobacterium tumefaciens, andcan be used as carriers for genetic transformation.(2) Tobacco Genetic transformation and detection of transgenic plant: Cotton chloroplastCu-ZnSOD gene plant expression vector was constructed and introduced into tobacco NC89 viaAgrobacterium tumefaciens. There 23 plant display positive reaction in kanamycin selection. The resultshowed that 4 plantlets display positive reaction in PCR analysis,Southern blotting and SOD enzymeactivity detection, also indicated that SOD enzyme activity in aparted cotyledon of 4 transgenic plantsis higher than of non transgenic plant, and the sensitivity to paraquat in non transgenic plant is higherthan in 4 transgenic plants. Totally, the integration of foreign gene increased tobacco resisted- caducityactivity.(3) Cotton genetic transformation: CCRI 24 and breeding material 652583 were used as materials for Agrobacterium infection, Cotton chloroplast Cu-ZnSOD gene plantexpression vector was constructed and introduced into CCRI 24 and breeding material 652583via Agrobacterium tumefaciens. There are 9 plantlet CCRI 24 display positive reaction inkanamycin selection, but resist seeding of breeding material 652583 is in selection still.