拟南芥脂类代谢在ER胁迫下的响应机制

Arabidopsis LEW1 encodes a cis-prenyltransferase, which catalyze the synthesis of dolichol, a lipid carrier of sugars used for protein glycosylation in endoplasmic reticulum (ER). Because of the damage of dolichol synthesis, the protein glycosylation of mutant lew1 was decreased so that lew1 was more sensitive to the inhibitor of protein glycosylation---Tunicamycin (TM). Ethyl methanesulfonate (EMS)-mutagenized M2 seeds of lew1 were screened for TM-insensitive mutants, and sol2 (suppressor of lew1 2) is the one, which is no longer sensitive to TM. Through map-based cloning and BSA sequencing, we found the mutation site of sol2 and cloned SOL2. According to previous research, SOL2 functions as a negative regulator of lipid synthesis in ER. We propose that lipid metabolism in ER may be regulated downstream of UPR (Unfolding Protein Response) pathway, and sol2 mutants survive in ER stress probably due to an accumulated lipid in ER, so that sol2 is not sensitive to TM.. Using the mutants of lew1, sol2 and UPR-pathway genes, the application aims to uncover the regulation of lipid metabolism in ER stress. By means of physiological analysis, cellular change, metabolic regulation and signal transduction, the regulation mechanism of UPR pathway in lipid metabolism under ER stress will be disclosed, providing the theoretical basis for breeding new crop varieties with high tolerance to stress and new rape varieties with high oil content.

拟南芥LEW1编码一个异戊二烯转移酶，催化内质网蛋白糖基化中多糖载体多萜醇的合成。突变体lew1中，LEW1活性降低，导致多萜醇合成受损、蛋白糖基化水平降低，因此突变体lew1对蛋白糖基化抑制剂TM敏感。对突变体lew1种子进行EMS诱变，课题组筛选到恢复突变体sol2，sol2突变体对TM不再敏感，甚至比野生型更抗TM。目前已通过图位克隆结合BSA测序找到sol2的突变位点。已有研究结果表明，SOL2作为负调控因子参与内质网脂类的合成。推测ER胁迫诱发的UPR参与脂类代谢的调控过程; sol2突变使内质网脂类合成水平提高，脂类积累有助于细胞适应ER胁迫，因此sol2对TM不敏感。项目将利用lew1、sol2及UPR信号途径的相关突变体材料，从生理、细胞、代谢及信号传导等方面开展研究，解析ER胁迫下脂类代谢调控机制，为培育抗性强的作物新品种及含油量高的油菜新品种提供理论基础。

本项目通过将对衣霉素(tunicamycin, TM)敏感的突变体lew1进行EMS重新诱变，从M2后代筛选得到一个对衣霉素不再敏感的突变体sol2。利用图位克隆的方法得到sol2的突变位点，互补实验验证了基因的功能。SOL2编码一个富含甘氨酸的小蛋白，参与脂类从内质网到叶绿体的运输。sol 2 突变导致细胞中三酰甘油（Triacylglycerols，TAG）积累量大大升高。转录组及脂质代谢组分析表明，sol2-1突变缓解了lew1突变造成的ER胁迫；培养基中添加游离脂肪酸使得lew1对TM敏感的表型得到部分恢复，而在sol2-1lew1背景下突变TAG水解酶SDP1使得其对TM又敏感了，这些结果都表明TAG的积累有助于植物适应内质网胁迫。同时，我们惊喜的发现内质网信号途径bZip28、bZip60及IRE1介导细胞自噬，通过细胞自噬来消耗脂质，其双突变体bzip28bzip60及ire1aire1b在内质网胁迫下TAG得到大量积累，而膜脂含量相应的有所降低，表明内质网信号途径通过消耗细胞中的TAG来满足内质网胁迫下对膜脂变化的需求。研究结果已申请国家发明专利2项，同时相关研究论文正在投稿中。