单胚RT-PCR:基因表达动态研究及应用技术探讨

为进一步探讨单胚RT-PCR技术的适用性,我们测试了3个特定的基因STM、MP和ASKη在拟南芥(Arabidopsisthaliana(L.)Heynh.)早期胚胎发生过程中的动态表达情况.进一步证实以该技术研究某些特定基因的表达动态是可行的.着重探讨了扩增产量与模板量的对应关系、基因组DNA的干扰以及DNase Ⅰ处理过程中的mRNA降解等主要技术问题.结果表明:材料制备时显微操作技术的稳定是确保单胚RT-PCR结果稳定的关键.应尽量缩短DNaseⅠ处理时间,以避免mRNA降解.

Single-Embryo RT-PCR:Application for Gene Expression Dynamics and Some Technical Aspects

An extended investigation was carried out for further demonstrating single-embryo RT-PCR technique, which was previously established in our laboratory as a useful tool on study of gene expression of Arabidopsis thaliana (L.) Heynh. Based on the previous work some important technical aspects in the application of the technique were mainly discussed in the present report. The dynamic expression of three selected genes, shoot-meristemless (STM), monppteros (MP) and Arabidopsis shaggy-like kinase etha (ASKh), during the embryogenesis were carefully compared. It was revealed that the technique was especially useful for tracing the expression dynamics of specific genes during embryogenesis. However, the quantity of starting material and the duration of DNaseⅠ treatment are critical points for successful application of the technique. The best condition for eliminating the possible influence of genomic DNA on data analysis was also tested and discussed.