论文标题:农杆菌介导花生白藜芦醇合酶基因转化马铃薯研究
Study on the Transformation of Potato with Peanut Resveratrol Synthase Gene Mediated by Agrobacterium Tumefaciens
论文作者
论文导师 陈由强,论文学位 硕士,论文专业 植物学
论文单位 福建师范大学,点击次数 731,论文页数 77页File Size8702K
2006-09-01论文网 http://www.lw23.com/lunwen_140297/
resveratrol; potato; vector construction; genetic transformation
利用质粒pCAMBIA1301构建以CaMV35S为启动子的花生白藜芦醇合酶基因表达载体pB3RS，通过PCR和限制性内切酶酶切验证构建正确。利用电穿孔法将重组质粒pB3RS导入根癌农杆菌菌株LBA4404中，并通过PCR和限制性内切酶酶切验证。以马铃薯克新3号脱毒种薯为受体材料，最佳分化培养基为MS+ZT 2.0 mg／L+IAA 1.0 mg／L，其愈伤组织诱导率为100％，不定芽分化率为80％。试验表明适宜的转化条件为：转化时外植体无需预培养，菌液OD_(600)=0.1-0.5，接种浸泡时间为5-10 min，共培养时间为2d。共获得10个目的基因PCR检测阳性的马铃薯无性系，对其中的克新3-2无性系进行Southern杂交鉴定，结果为阳性，表明外源目的基因可能已整合到植物基因组中。
The expression vector pB3RS with the peanut resveratrol synthase gene (Genbank AY170347) driven by CaMV35S promoter was constructed on the basis of pCAMBIA1301 and identified by PCR and enzyme digestion. The vector pB3RS was transferred into the Agrobacterium tumefaciens strain LBA4404 by electro-transformation and identified by PCR and enzyme digestion. The optimal redifferentiation medium for explants of virus-free potato tubers of "Kexin 3" was MS + ZT 2.0 mg/L + IAA 1.0 mg/L. The callus induction rate on the medium was 100% and the adventitious shoot redifferentiation rate 80%. The test showed that the pre-culture of explants of virus-free potato tubers of "Kexin 3" was not necessary. Using OD_(600) 0.1-0.5 solutions of A. tumefaciens LBA4404/pB3RS, submerging explants in bacterium solutions for 5-10 min and co-culturing explants for 2d with the bacteria was suitable for genetic transformation of "Kexin 3". 10 potato clones showed a fragment identical with RS gene in PCR analysis. One of them, "Kexin 3-2" clone, was selected for southern blot analysis and the result was positive in the clone but negative in the control.
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