通过遗传转化获得含多基因的水稻转基因纯合植株

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（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot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，鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot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同时改良作用多个性状提供了一种途径。

The Development of Transgenic Rice Homozygous Plants Containing Multiple Genes via

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.