农杆菌介导的水稻草矮病毒NS6基因的转化

水稻草矮病毒(Ricegrassystuntvirus,RGSV)RNA6片段毒义链编码的非结构蛋白NS6,与病害症状密切相关,被称为病害特异蛋白.因此,应用农杆菌介导法,选取对RGSV表现不同抗性的台农67、中花6号、中花12号、中花15号、台中1号、合系28和063817个水稻品种,以其未成熟胚或成熟胚预培养4d后,诱导生长旺盛的愈伤组织为外植体,将NS6基因导入其中,对影响水稻再生及农杆菌转化的主要因素进行了比较研究,并获得了转NS6基因工程植株.结果表明,以未成熟胚为受体,获得的抗性愈伤组织转化率明显高于成熟胚;水稻不同品种对农杆菌转化反应不同;添加一定浓度乙酰丁香酮和葡萄糖,可提高抗性愈伤组织形成率;选用G418进行抗性筛选能获得转NS6基因再生植株,用卡那霉素筛选产生的愈伤组织则未能获得再生植株.

Agrobacterium-mediated transformation of NS6 gene of Rice grassy stunt virus

The non structural protein encoded by Rice grassy stunt virus (RGSV) RNA6 vRNA was found in large amount in infected rice tissues. Therefore the plant expression vector pCBTNSv6 harboring NS6 gene of RGSV was transformed into the calli of rice by Agrobacterium mediation. After selection and regeneration, transgenic plants were obtained. PCR and southern blotting analysis showed that the NS6 gene was integrated into the rice genome. By Agrobacterium mediated method, NS6 gene was transformed into varieties, which were different in resistance to RGSV. The methods were optimized, and the important factors which influence transformation including tissue culture condition, variety explants were studied. Immature embryos of different rice varieties precultured for 4 d were infected and transformed with Agrobacterium LBA4404. The resistant calli were transferred into the differentiation medium and transformed plants were regenerated. The efficiencies of transformation and transgenic plant regeneration varied greatly with varieties. Adding acetosyringone (As) into co cultivation medium could raise the transformation frequency. The stable transformation efficiency reached 35%, which was measured as numbers of Geneticin (G418) resistant calli produced. Some G418 resistant plants were obtained, but no Kan resistant plant regeneration was observed.