AcSERK1基因在菠萝体细胞胚发生初期特异性表达的分子机制

Pineapple (Ananas comosus (L.) Merr.） a perennial herbaceous fruit tree, is widely-spreaded and cultivated in tropical region. It will be of great importance for the genetic improvement and industrial breeding of pineapple to study the molecular mechanism of its somatic embryogenesis (SE) regulation. SE starts with the conversion from somatic cell to embryogenic cell. During this transition phase, a stage-specifically expressing gene, AcSERK1 played a key role in signal transduction. However, there is no research reporting about the molecular mechanism of its expressiong regulation. Our previous works showed that AcSERK is a gene family consisting of 3 members. Only AcSERK1 participates in induction of SE, and its 5′ upstream region between nt -1137 /-722 was supposed to possess embryogenic cell specific regulatory function. Besides, the sub-sequences between nt -227 / -1 had the basal transcriptional activity, which contains 2 CpG islands, these two regions interacted with each other to play an important role to started the stage-specific expression pattern of AcSERK1 during the early of SE in the pineapple. Based on these results, the present project will isolate and identify the embryogenic cell specific cis-element and then select and characterize the transcription factor which interact with the specific cis-element . In addition, we will also analyze the impact of different methylation levels of AcSERK1 5'-upstream region on its transcription activity. Summary above, we would clarify the molecular mechanism undelying regulation of AcSERK1 gene during the early of SE, expecting to what metioned above will be the foundation of further study for analyzing transcription factors and afford an efficient way to regulate the somatic embryogenesis.

菠萝是多年生热带草本果树，深入研究其体细胞胚发生的分子调控机理对推动工厂化育苗有着重要意义。体细胞胚发生始于从体细胞到胚性细胞的转变，而 SERK基因的特异表达在这一过程中起着重要的信号转导作用，但国内外尚未见对其表达调控分子机制的报道。本课题组研究表明，菠萝有3个SERK基因家族成员，只有AcSERK1基因参与了体细胞胚诱导，其 5`上游-1137/-722区具有胚性细胞特异性调控功能、-227/-1区具有基础转录活性，两个区域配合启动AcSERK1基因在菠萝体细胞胚发生初期发生大量表达， 5`上游区存在2个CpG岛。本申请将在上述基础上，继续分离AcSERK1基因的胚性细胞特异性序列、筛选和鉴定与该序列互作的转录因子、分析5`上游甲基化对转录的影响，解析该基因在菠萝体细胞发生初期特异性表达的分子机制，为进一步研究该基因表达调控网络和有效调菠萝体细胞发生打下基础。

菠萝是多年生热带草本果树，深入研究其体细胞胚发生的分子调控机理对建立高效的转化受体系统和推动工厂化育苗有着重要意义。课题组前期的研究表明：菠萝体细胞胚发生始于从体细胞到胚性细胞的转变，而AcSERK1基因的特异表达在这一过程中起着重要的信号转导作用，其5'上游-1137/-722nt区具有胚性细胞特异性调控功能。. 通过对AcSERK1 5'上游调控序列 (-2090 /+258 nt)进行了一系列的缺失分析，确定位于其5'上游-921 / -881 nt区段为胚性细胞特异性表达调控区，从而将具有胚性细胞特异性调控功能区段从426bp(-1137/-722nt ) 进一步缩短至长度为41bp（-921 / -881 nt）。. 以 -921 /-881 nt构建诱饵载体pAbAi-SE41，采用酵母单杂交技术从菠萝胚性细胞cDNA文库中筛选获得了AcHB13和AcKAN2等2个转录因子。在超表达的转基因株系中，这2个基因表达量和体细胞胚发生量也显著提高。. 对AcHB13、AcKAN2及-921/-881nt区的相互关系的一系列分析结果显示：2个转录因子分别通过结合-921/-881nt区中的CCCATA和AATTAGAC两个顺式元件，激活启动子活性，调控AcSERK1的表达；且AcHB13和AcKAN2蛋白之间存在互作关系，形成二元复合物行使功能。.AcSERK1转录起始位点附近的2个CpG岛在胚性细胞中发生甲基化的位点个数显著少于非胚性细胞；用甲基化抑制剂使AcSERK1 5'上游去甲基化修饰后，能提高其表达量，促进体细胞胚的发生。. 因此，AcSERK1胚性细胞特异性调控功能区位于-921 / -881 nt，其特异表达受AcHB13和AcKAN2调控，而其转录起始位点附近的甲基化会影响其表达量。该研究结果为进一步解析该基因表达调控网络和有效调菠萝体细胞发生提供了依据。