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采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。
Agrobacterium tumefaciens strain GV3101 was used to infect alfalfa somatic embryos to study the genetic transformation of alfalfa somatic embryos. Agrobacterium strain GV3101 biphasic vector pCAMBIA2301, which has a gus reporter and an nptII anti-kanamycin screening gene. Infected cotyledons of alfalfa somatic cells in 75 mg / L kanamycin screening pressure, after a series of induction culture, the final access to transgenic plants. GUS expression in different organs of the transgenic plants was then examined by GUS histochemical localization analysis and further the stable integration and conversion of transgenes were confirmed by PCR and Southern blotting. The results showed that GUS was expressed in different organs of transgenic plants, the copy number of integrated npt Ⅱ gene was 1 ~ 4, and the transformation rate of transgenic plants obtained was 65.82%.