Regulation of gene expression during conifer embryogenesis

利用体细胞胚胎发生(SE)进行无性系繁殖可以在更短的时间内将优良材料输送到商品林，并与有性过程相结合用于种子生产，有助于维持种子园的正确遗传平衡。目前，在针叶树中成功地引入商业胚胎发生的制约因素被认为是：a)提高胚胎质量；b)效率和响应基因类型的数量；c)从成熟组织中诱导SE。为了开发白云杉和道格拉斯冷杉这两个加拿大重要经济树种的SE，我们一直在研究SE和ZE发育过程中的分子事件。对成熟过程中表达的cDNA和基因组序列的分析为针叶树胚胎成熟的分子生物学提供了重要的信息，并为后续的研究奠定了基础，这些研究将集中在胚胎发育的非常早期阶段。我们将利用同源的胚胎调控探针(转录因子PmLEC1和PmCOP1，这是在道格拉斯冷杉和SE早期表达的两个关键基因)和DD-PCR来研究自花授粉和异花授粉种子早期胚胎发生的分子基础、MG、胚柄和其他因素。这些基因的功能分析将通过反向遗传学进行。此外，还将检查E和NE培养在诱导阶段的差异，以了解基因产物在SE启动中所起的作用。针叶树E培养物的形成依赖于基因型，并且诱导频率在不同家系之间可能有很大的差异。方法包括基于聚合酶链式反应的SSH基因文库结合微阵列和/或基于定量RT-PCR的图谱分析。基于微阵列的方法将允许同时定量大量转录本，以确定在SE启动和响应期间的协调基因组(S)，包括油菜素内酯处理导致包括道格拉斯冷杉在内的各种类型针叶树SE频率增加。本研究具有基础性和实践性，对HQP的培训也具有重要意义。

Clonal propagation by somatic embryogenesis (SE) has the potential for the delivery of superior material to the commercial forest in a much shorter time and used in combination with the sexual process for seed production will help maintain the correct genetic balance from seed orchards. Presently constraints to the successful introduction of commercial embryogenesis in conifers are perceived to be: a) improvement of embryo quality; b) efficiency and the number of responding genotypes; c) induction of SE from mature tissues. To develop SE for white spruce and Douglas fir, two economically important species in Canada, we have been examining the molecular events during development of SE and ZE. The analysis of cDNA and genomic sequences expressed during maturation have provided important information on molecular biology of conifer embryo maturation and form the basis of continuing proposed studies that will focus on very early stages of embryogeny. The molecular basis of early embryogeny, role of MG, suspensor, and other factors in self- and cross-pollinated seeds will be studied using the homologous embryo regulatory probes (transcription factor PmLEC1, and PmCOP1, two critical genes expressed during early Douglas fir ZE and SE, (isolated in my lab), and DD-PCR. The functional analysis of these genes will be performed by reverse genetics. In addition, differences between E and NE cultures during the induction phase will be examined to understand what roles the gene products play in initiation of SE. Formation of conifer E culture is genotype dependent and induction frequencies can vary widely among families. Approaches include PCR based SSH cDNA libraries coupled with microarray and/or quantitative RT-PCR based profile analyses. Microarray based methodology will allow the simultaneous quantification of a large number of transcripts to identify coordinated gene groups during SE initiation and response (s) to PGRs including Brassinolide treatment implicated in increased SE frequency from a wide range of genotypes of conifers, including Douglas fir. The research is of fundamental and practical interest and will also be important in training of HQP.