通过遗传转化获得含多基因的水稻转基因纯合植株(英文)  

The Development of Transgenic Rice Homozygous Plants Containing Multiple Genes via Genetic Transformation

作　　者：唐克轩[1] 孙小芬[1] 姚剑虹[1] 戚华雄[2] 卢兴桂[2] 

机构地区：[1]复旦大学生命科学学院遗传工程国家重点实验室,上海200433 [2]湖北农科院杂交水稻研究中心,武汉430064

出　　处：《复旦学报（自然科学版）》2001年第5期483-490,496,共9页Journal of Fudan University：Natural Science

基　　金：Supported by the Rockefeller Foundation, USA (Grant No. RF97001 #594);Supported by China Na-tional High-Tech "863" program (G

摘　　要：利用基因枪法将含有４个不同基因的３个质粒共转化由粳稻品种鄂宜１０５号和鄂晚５号种子胚　诱导的愈伤组织（５～１０　ｄ龄）．　从轰击的９８６块愈伤组织中共再生出１６９株独立的转基因水　稻植株（转化率为１７％）．　ＰＣＲ／Ｓｏｕｔｈｅｒｎｂｌｏｔ分析显示７０％以上的转基因植株含有　所有４个基因．　ＧＵＳ组织化学分析、Ｗｅｓｔｅｒｎ　ｂｌｏｔ和／或ＲＴ－ＰＣＲ分析表明所有４个基因的共表　达率为７０％．　未观察到任何质粒在整合中存在优势，转基因拷贝数也与基因表达量无关．　遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传．　从其Ｒ１代为３∶１孟德尔方式遗传　的后代Ｒ２代植株中，鉴定出含有３个或４个不同基因的转基因纯合植株系．　ＰＣＲ／Ｓｏｕｔｈｅｒｎ　ｂｌｏ　ｔ分析证实了这些转基因纯合植株系．　这些系的植株具有相似的外源基因表达量．　我们证实　通过基因枪介导的共转化，结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株　．　这项技术为利用基因工程同时改良作物多个性状提供了一种途径．Five to ten d-old mature seed-derived callus from 2 elite japonica rice cvs. Eyi 105 and Ewan 5 was cotrans- formed with three plasmids containing four different genes via particle bombardment. One hundred and sixty-nine inde- pendent transgenic rice plants were regenerated from 986 bombarded calli with a transformation frequency of 17%. Over 70% of transgenic plants contained all the four genes, revealed by PCR/ Southern blot analysis and a 70% co-ex- pression frequency for all the four genes was obtained, indicated by GUS histochemical assays, western blot and/or RT- PCR analysis. No preferential integration of any specific plasmid or correlation between transgene copy number and lev- els of gene expression was observed. Genetic analysis confirmed Mendelian segregation (3: 1 ) of most transgenes in progeny. Five independent homozygous plants containing three or four different transgenes were identified from R2 generations with their R1 parent plants showing 3: 1 Mendelian segregation patterns. Their homozygous status was confirmed by PCR/Southern blot analysis. The homozygous plants also exhibited similar levels of transgene expression. It is demonstrated that homozygous transgenic rice plants containing multiple genes can be obtained via particle bom- bardment-mediated co-transformation and through conventional breeding selection based on genetic analysis. This tech- nique offers the way for the gene engineering of multiple traits in crop plants.

关 键 词：转基因水稻 分离分析 转基因纯合植株系 遗传转化 基因工程 基因表达 孟德尔方式遗传 

分 类 号：S511[农业科学—作物学] S188