Yang Guojun, Lee Yeon-Hee, Jiang Yiming, Kumpatla Siva P, Hall Timothy C
Institute of Developmental and Molecular Biology and Department of Biology, Texas A and M University, College Station, TX 77843-3155, USA.
Plant Mol Biol. 2005 Jun;58(3):351-66. doi: 10.1007/s11103-005-5101-y.
Despite the presence in nature of many functional gene families that contain several to many highly similar sequences, the presence of identical DNA sequence repeats is widely thought to predispose transgene inserts to homology dependent gene silencing (HDGS). The induction of transcriptional gene silencing (TGS) by RNAs homologous to promoter sequences has been reported recently in Arabidopsis and humans. However, mechanisms for TGS have not been studied in detail for rice, the most widely cultivated crop plant. Taking advantage of a well-characterized homozygous silenced transgenic rice line (siJKA), supertransformation was performed with a binary vector bearing mUbi1 and 35S promoter sequences identical to those in the resident transgenes. Analysis of the incoming and resident transgenes in the supertransformants revealed that the incoming mUbi1 transgene promoter was not silenced whereas the incoming 35S transgene promoter was silenced. That the resident silenced mUbi1-bar was not reactivated in these experiments as a result of passage through tissue culture and regeneration was established by the finding that regenerants from siJKA immature embryos were all silenced for mUbi1-bar. In a parallel experiment, when wild type rice calli were transformed with the same binary vector, neither of the incoming transgene promoters was silenced. Following 5-azacytidine (5-azaC) treatment of siJKA, aberrant RNA species corresponding to the 35S promoter, but not to the mUbi1 promoter, were detected. Nevertheless, no 21-25 nt RNAs corresponding to the 35S promoter sequence were detected. These results, together with detailed analyses of the progenies from the primary transformants and supertransformants, revealed that HDGS of the resident silenced locus was caused not by simple transgene duplication, but by aberrant transcripts derived from rearranged promoters present in siJKA. Practical consequences of this study include a justification for the use of multiple copies of a given promoter for transformation without inducing silencing, provided that their genomic integration does not result in aberrant transcription of the promoters.
尽管自然界中存在许多功能基因家族，其中包含几个到许多高度相似的序列，但人们普遍认为，相同DNA序列重复的存在会使转基因插入易发生同源依赖性基因沉默（HDGS）。最近在拟南芥和人类中报道了与启动子序列同源的RNA诱导转录基因沉默（TGS）。然而，对于种植最广泛的作物水稻，尚未对TGS的机制进行详细研究。利用一个特征明确的纯合沉默转基因水稻系（siJKA），用携带与常驻转基因中相同的mUbi1和35S启动子序列的二元载体进行了超级转化。对超级转化体中导入和常驻转基因的分析表明，导入的mUbi1转基因启动子未被沉默，而导入的35S转基因启动子被沉默。通过发现来自siJKA未成熟胚的再生植株中mUbi1-bar均被沉默，证实了常驻沉默的mUbi1-bar在这些实验中未因组织培养和再生而重新激活。在一项平行实验中，当用相同的二元载体转化野生型水稻愈伤组织时，两个导入的转基因启动子均未被沉默。用5-氮杂胞苷（5-azaC）处理siJKA后，检测到与35S启动子相对应的异常RNA种类，但未检测到与mUbi1启动子相对应的异常RNA种类。然而，未检测到与35S启动子序列相对应的21-25 nt RNA。这些结果，连同对初级转化体和超级转化体后代的详细分析，表明常驻沉默位点的HDGS不是由简单的转基因重复引起的，而是由siJKA中重排启动子衍生的异常转录本引起的。这项研究的实际意义包括，只要给定启动子的基因组整合不会导致启动子的异常转录，就可以合理地使用多个拷贝的启动子进行转化而不诱导沉默。
