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

甘蓝型油菜小孢子的有效胚胎发生已经建立起来。该体系对发育研究和实际育种中纯合品系的生产具有重要的示范意义。为了深入了解油菜小孢子胚胎发生的分子机制，本研究从油菜小孢子胚胎发生的早期阶段分离了与胚胎发生诱导相关的基因，分析了一个胚胎发生特异的启动子P22a1，并开发了收集油菜胚性小孢子的有用工具。1.利用抑制消减杂交技术从油菜小孢子胚胎发生的早期分离到136个基因。对这136个基因进行BLASTX同源检索，发现87个基因与已知基因同源，23个基因与未知基因同源，26个基因在数据库中没有mach。从这136个基因中筛选出15个基因进行实时荧光定量RT-PCR检测，发现有14个基因在胚胎发育早期高表达。启动子肛门…对拟南芥同源基因的进一步分析表明，这些基因分为两类：(1)仅在小孢子胚胎发生中表达的基因，而在合子胚胎发生中不表达的基因；(2)在两种胚胎发生中共同表达的基因。2.将胚胎发生特异性启动子P22a1与GUS和GFP报告基因融合，并导入拟南芥和油菜中，检测该启动子的时空特异性。在受精前的幼胚囊、受精卵、幼胚和胚乳细胞中观察到特异性的GFP积累。在启动子-353~-249之间发现了一个顺式作用增强子样区，但除了104by区外，还需要其他顺式作用元件(S)才能实现完整的启动子活性。从转P22a1：：GFP基因油菜植株中分离出的小孢子在培养36小时内形成了表达GFP的原胚性细胞。结果表明，启动子：：报告基因可能是一种有用的工具，用于浓缩在形态分化之前将发育过程从孢子发生转换到胚胎发生的细胞。较少

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