农杆菌介导转化法构建转CTV-cp的枳壳植株

目的 :采用农杆菌介导法 ,将能抑制病毒侵染的外壳蛋白基因转入枳壳 ,为通过基因转化进行枳壳的抗病育种提供依据和方法。方法 :以枳壳实生苗上胚轴为材料 ,农杆菌菌株为EHA₁₀₁,含有质粒载体 pGA482GG。质粒上插入了 35s启动子驱动的外源目的基因———柑桔衰退病病毒外壳蛋白基因 (CTV cp)及GUS ,NPTⅡ基因。结果 :枳壳转化试验中 ,抗菌素采用头孢霉素较好 ,浓度为 300mg·L^-1;以卡那霉素作为选择试剂 ,浓度为 5 0mg·L^-1;转化获得的抗性植株 ,GUS检测结果 ,70.0%的植株呈阳性反应 ;Southern杂交分析 (Southernblot)鉴定证明外壳蛋白基因已整合到枳壳植株的核基因组中。结论 :通过农杆菌介导的转化法 ,成功地获得了转外壳蛋白基因的枳壳植株。

Plant Regeneration from Agobacterium-mediated CTV-cp Gene Transformation of Poncirus trifoliata Raf.

Objective:To lay a foundation for the resistant breding, the anti virus CTV cp gene was transformed into the epicotyles mediated by Agrobacterium tumefaciens in Poncirus trifoliata . Method:The explants used for the genetic transformation were the epicotyls from P.trifoliata. The Agrobacterum tumefaciens strain was EHA₁₀₁ containing vector plasmid pGA482GG.The coat protein gene (CTV cp gene),GUS gene and NPT II gene were introduced into the transformation plasmid.Results:Ceftaxime used as antibiotics was better than carbenicillin. The concentration was 300 mg·L^-1 ;The selection pressure for kanamycin was 50 mg·L^-1 ;70.0% of the resistant plants were GUS positive; extra gene was proved to be in P.trifoliata plant by southern blot examination. Conclusion:An effective genetic transformation mediated by Agrobacterium tumefacines, which harbours a CTV cp gene, has been developed in P.trifoliata. Transgenic CTV cp plants were obtained.