一种快速高效的水稻原生质体制备和转化方法的建立

在模式植物拟南芥中，原生质体瞬时表达技术已被广泛地应用到功能基因组学的研究中，但水稻原生质体因其制备过程相对繁琐，转化效率偏低，尚未在基因功能研究中获得广泛应用。本研究在拟南芥原生质体制备和转化的基础之上，对水稻原生质体的制备和转化方法进行改良优化。以水稻幼茎为起始材料，采用纤维素酶R-10和果胶酶R-10，对水稻组织进行消化并利用蔗糖密度梯度自沉降的方法分离原生质体，获得了高纯度的原生质体。对质粒转化原生质体时的转化方法、转化时间及质粒浓度进行探索，在缩短原生质体分离时间的同时，大大提高了转化效率。用较少量的质粒DNA即可获得外源基因在原生质体内高效的表达，且转化效率可达70％。我们建立的这种快速有效的水稻原生质体制备和转化方法，可为水稻功能基因组学研究提供技术支持。

A Rapid and Efficient Method for Isolation and Transformation of Rice Protoplast

Transient gene expression in protoplast is a common approach for studying subcelluar localization, promoter activities and protein complexes in model plant, Arabidopsis. However, protoplast isolation, transformation and as well as downstream analyses in rice are often hampered by a number of factors such as time-consuming, cost-intensive and low transformation efficiency. In this study, we reported a rapid and efficient method for isolation and transformation of rice protoplast, based on the recently published procedure for Arabidopsis protoplast preparation. 10-to-14-days stem tissues but not leaf tissues were digested by proper cellulase R-10 and mecerozyme R-10. Pure protoplasts without cell debris were isolated from the interphase of sucrose gradient after natural sink. This method was also very time-efficient, large amount of protoplasts could be obtained within one day. The transformation efficiency was pretty high （70%） with little amount of DNA. The method we presented here should be valuable for the functional genomics research in rice.