摘要
Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.
[目的]探索籼稻品种Kasalath的遗传转化条件,为针对该品种进一步的分子生物学研究奠定基础。[方法]以籼稻品种Kasalath的愈伤组织为转化受体,应用根癌农杆菌介导法进行遗传转化;从共培养方式、共培养时间、共培养后处理方法等方面优化遗传转化体系。①共培养条件的优化:愈伤组织接入共培养基时分2组,1组加1层灭菌滤纸,1组不加。②共培养时间的优化:分别于农杆菌液侵染愈伤1,2,3,4 d观察。③共培养后处理方式的优化:方式1,将共培养后的愈伤组织用灭菌蒸馏水冲洗多次至水清亮无混浊悬浮物,然后用含有羧苄青霉素的灭菌蒸馏水浸泡30 min,放置在有3层滤纸的灭菌培养皿中短暂干燥。方式2,将共培养后的愈伤组织置于带2层滤纸的灭菌培养皿中干燥,干燥3 d后取出。2种方式干燥后的愈伤组织均接入含40 mg/L潮霉素筛选压的筛选培养基上,筛选2次,每次2～3周。[结果]Kasalath的愈伤组织经农杆菌侵染后,经过共培养,再经筛选培养出抗性愈伤直至分化成转化苗,共培养时问对于转化效率的影响较大。如果共培养时间过长,将导致农杆菌过度生长,随后的筛选培养中即使加入抗菌的羧苄青霉素也无法抑制其生长,愈伤组织褐化死亡率增大。而共培养时间太短,则影响农杆菌Ti质粒T-DNA的转移,影响转化效率。该研究中当愈伤组织与农杆菌共培养时间为2 d时的转化效果最好,抗性愈伤组织获得率达84.1%,转化菌的转化率达到73%,且共培养时愈伤组织是否直接接触培养基对转化无明显影响。利用PMCG161载体引物PMCGF和PMCGR对所获得的部分转化苗进行PCR检测,23株转化苗中有13株扩出了预期的约750 bp条带,且为阳性转化苗,表明外源基因OsMAPK2已整合到水稻品种Kasalath的基因组中。[结论]经愈伤组织的诱导、共培养、筛选、预分化、分化等步骤,最终成功实现了农杆菌介导的OsMAPK2基因对籼稻品种Kasalath的遗传转化。
基金
Supported by Youth Foundation of National Natural Science(30600400)
Chenguang Program of Youth Science and Techonogyof Wuhan City(00750731302)
Introduced Talents Started Projectof South-Central University for Nationalities(YZZ05012)~~
