Analysis of microspore embryogenesis by exhaustive isolation of cDNA and development of useful tool collecting embryogenic microspores

在芸苔属植物中已经建立了有效的小孢子胚胎发生。这一系统在发育研究以及在实际育种中产生纯合系方面具有重要的模式意义。为了深入了解油菜小孢子胚胎发生的分子机制，本研究尝试分离与小孢子胚胎发生相关的基因，分析油菜小孢子胚胎发生特异启动子P22 a1，并建立一种有效的小孢子收集工具。对这136个基因进行BLASTX同源性搜索，发现87个基因与已知基因同源，23个基因为同源的未知基因，26个基因在数据库中没有发现。从这136个基因中筛选出15个基因，通过实时荧光定量RT-PCR检测其表达水平，其中14个基因在胚胎发生早期就表现出高表达。肛门启动子 ...更多信息 对拟南芥同源基因的分析表明，这些基因可分为两类：（1）仅在小孢子胚胎发生中表达，而在合子胚发生中不表达的基因;（2）在两种胚胎发生中都表达的基因。将胚胎发生特异性启动子P22 a1与GUS和GFP报告基因融合，导入拟南芥和油菜中，研究启动子P22 a1的时空特异性。GFP在受精前的幼胚囊、合子、幼胚和胚乳细胞中均有特异积累。在启动子-353至-249处鉴定出顺式作用增强子样区域，但是除了104 by区域之外，其他顺式作用元件对于完整的启动子活性是必需的。从P22 a1：：GFP转基因油菜植株中分离的小孢子在培养起始后36小时内形成表达GFP的原胚细胞。结果表明，启动子：：报告基因将是一个有用的工具，集中细胞，他们的发育过程中转换从孢子发生到胚胎发生之前，他们可以在形态上区分。少

Effective embryogenesis from isolated microspores has been established in Brassica spp. This system has great importance as a model in developmental study as well as in production of homozygous lines in practical breeding. To understand molecular mechanism underlying microspore embryogenesis, we have attmpted to exhaustively isolate genes related to induction of embryogenesis, to analyze an embryogenesis-specific promoter P22a1, and to develop a useful tool collecting embryogenic microspores in rapeseed.1.The 136 genes were isolated from early stage of microspore embryogenesis of rapeseed by suppression subtractive hybridization. BLASTX homology search for these 136 genes revealed that 87 genes were homologous to known genes, 23 ones were homologous unknown genes, and 26 ones had no mach in the database. When 15 genes selected from these 136 genes were examined their expression level by real-time RT-PCR, 14 genes showed the high expression in early stage of embryogenesis. Promoter anal … More ysis using Arabidopsis homologues indicated that the genes were classified into two types, (1)genes expressed in only microspore embryogeneis, but not in zygotic embryogenesis, (2)genes commonly expressed in both embryogenesis. The genes classified in former type are considered to be candidate genes related to induction of androgenesis.2.An embryogenesis-specific promoter P22a1 was fused to GUS and GFP reporter genes and introduced into Arabidopsis and rapeseed to examine spatial and temporal specificity of the promoter. The specific GFP accumulation was observed in young embryo sacs before fertilization, zygotes, young embryos and endosperm cells. A cis-acting enhancer-like region was identified in the promoter from -353 to -249, but other cis-acting element(s) would be necessary in addition to the 104 by region for complete promoter activity. Microspores isolated from P22a1 : : GFP transgenic rapeseed plants developed GFP-expressing pro embryonic cells in 36 hrs after culture initiation. The result suggested that the promoter : : reporter gene would be a useful tool to concentrate cells that switched their developmental course from sporogenesis to embryogenesis before they could be morphologically distinguished. Less