Construction Of Plant Expression Vector With Rip Gene Of Barley And Its Transferring Into Rice

Rice is one of the most important crops,but the yield is declined every year because of its fungi disease. One of the common protecting method is chemical medicine. Although it has some effect,it also brings some disadvantages,such as heavy polluted environment,maladjustment ecology and harm to human healthy. It is testify that culture the fungi-resistant cultivates is the most economic and available way. However,the conventional breeding can't meet the need of cultivating the fungi-resistant varieties,because of its long period,short fungi-resistant genes and disappearing resistance with the changes of pathogen. So,using the genetic engineering to breed the fungi-resistant varieties is a available method.In this study,we constructed two plant expression vector with rip gene from barley and linked to two promoters which have high promoting efficiency in monocotyledonous plants,respectively,and then transferred to rice mediated by Agrobacterium Tumefaciens. The main factors of affecting transformation were investigated and established its regeneration and transformation systems. PCR analysis showed primarily that rip gene had already integrated into rice. The main results was summarized as follows.1 Constructed plant expression vectors with rip gene of barleyWe constructed four vectors altogether and two was intermediate vector (pUR and pJAR),The other two was plant expression vectors (pCAR and p3301RIP).(1)The intermediate vector pJAR harbors ActI promoter,regulating rip gene,and bar geneas selectable marker. This vector was used in gene gun transformation. (2)The interest gene in pCAR is under the control of ActI which is the strong promoter inrice. The selectable marker gene is hyg gene. (3)The interest gene in p3301RIP is under the control of double 35S promoter and theselectable marker gene is bar gene.2 Established the regeneration system with mature embryos of rice(1)The optional inducing medium of callus is MS basal medium supplemented 3mg/L 2,4-D,30g/Lsucrose and 8g/L agar.(2)The rate of callus induction varied with the genotypes. Dongnong V- 10 has the highest inducing rate among 4 tested genotypes,up to 96.1%;The second is Song 93-8 and Dongnong 419,and the average inducing rate renched 88.9% and 88.3% respectively.Fushiguang has the lowest inducing rate,up to 78.9%.(3)The optional subculture medium is MS basal medium with 2mg/L 2,4-D,40g/L sucrose and lOg/L agar. High sucrose and agar is apt to keep the tight close structure of callus.(4)The optional inducing medium of adventitious buds is MS basal medium with 2mg/L 6-BA,30g/L sucrose and 9g/L agar. All the tested genotypes have high inducing rate on this medium. The inducing ratio as follows:Song93-8 962.9%,DongnongV-10 679.7%,Dongnong -419 542.5%,Fushiguang 482.6%,which has significantly difference compared to other treatments.(5) The inducing ratio of adventitious buds has difference among genotypes. Song93-8 has the highest average inducing rate,up to 608.1%,and then DongnongV-10 up to 386.9%,then Dongnong419 up to 268.0%. Fushiguang has the lowest inducing,up to 202.2%.3 established transformation system mediated by Agrobacterium Twnefaciens.(1) The selection concentration of PPT is 10mg/L(2) The selection concentration of Hgy is 50mg/L(3) The better combination of normal experiments which influence the 3 factors of transformation showed as follows. The suspend liquid of Agrobacterium Twnefaciens is YEB;The pH in co-cultivation medium is 5.2;Hydroxybenzene subsidiary is trans-Ferulic acid. The order of affecting factors as follows:hydroxybenzene subsidiary>suspend liquid>pH.4 Obtained transgenic plants with rip gene verified by PCR.PCR analysis showed rip gene has integrated into rice genome primarily. The positive tran-sgentic plants with LBA4404(p3301RJP) is DongnongV-10 2 and Fushiguang 2. The positive transgentic plants with LBA4404(pCAR) is Song93-8 3 and Fushiguang 1.Postgraduate:CuiHailan Major :Botany Supervisor :Prof.Zhu Yanming...