转mCherry基因水稻的遗传分析及T-DNA整合位点的研究

Genetic Analysis of mCherry Transgenic Rice and Molecular Characte-rization of T-DNA Integration Sites in the Rice Genome

为了建立转基因水稻的高通量遗传分析系统,本研究以粳稻成熟胚诱导的愈伤组织为受体材料,潮霉素磷酸转移酶(HPT)为抗性筛选标记,通过农杆菌介导的方法将红色荧光蛋白基因mCherry导入水稻。在水稻转化愈伤组织、转化植株(T0)和转基因后代(T1)均检测到红色荧光,证实mCherry在转基因水稻中稳定表达。139个独立转化株系的遗传分析表明,mCherry在转基因后代表现多种遗传模式,54%的转基因株系呈单位点遗传。TAIL-PCR进一步验证转基因整合到水稻基因组。根据T-DNA整合位点DNA序列分析结果,T-DNA左边界发生碱基缺失、增加和替换,右边界只发生碱基缺失,说明左边界比右边界有更多的碱基变异类型。此外,对T2代植株mCherry和HPT的转录水平进行实时定量逆转录PCR分析,结果表明,mCherry和HPT转录水平因T-DNA在水稻基因组中的整合位置而变化,表现位置效应。本研究在mCherry荧光蛋白和其它相关方法的基础上,为转基因水稻遗传及分子分析建立了一个高效的流程体系。

To establish a high-throughput system for genetic analyses of transgenic rice, the red fluorescentprotein, mCherry, was introduced into japonica rice via Agrobacterium mediated transformation using matureembryo derived rice callus and the hygromycin phosphotransferase （HPT） selectable marker. Red fluorescence wasdetected in transformed rice callus, primary transformed plants （To） and T1 progenies, indicating stable expressionof mCherry in transgenic rice. Genetic analysis of 139 independent transformed rice lines indicated that mCherryshould be inherited in the transgenic progenies with multiple patterns, and 54.0% of the transgenic lines showedsingle-locus inheritance. Transgene integration in the rice genome was further confirmed using TAIL-PCR.Sequence analysis of T-DNA integration sites showed that nucleotide deletion, insertion and substitution occurredat the T-DNA left border, whereas the nucleotide deletion only happened in the T-DNA right border, whichsuggested that the left border region should have far more diverse types ofnucleotide variation than the right borderregion. Furthermore, real-time reverse-transcription PCR of mCherry and HPT in T2 transgenic plants revealedpositional effects, of which the mCherry and HPT transcript levels varied in the transgenic lines depending onT-DNA integration locations in the rice genome. Overall, the system based on mCherry and other related methodsdescribed in this study presented a high-efficient streamline for genetic and molecular analysis oftransgenic rice.