羊草新基因LcFIN2和LcFIN3的抗低温功能研究

Leymus chinensis (sheepgrass) is an important forage species in China and is known for its strong resistance, and can survive under freezing temperatures -47.5℃. Discovering the genetic resources of sheepgrass is important for understanding the molecular mechanisms of stress resistance in sheepgrass. In previous study, two novel genes LcFIN2 and LcFIN3 were cloned. The novel genes and its homologs produce hypothetical proteins with no conserved functional domains, and the genes products were mainly localized to the chloroplast. LcFIN2 showed a lower homology with other species, and LcFIN3 showed a high homology with predicted protein products of Oryza sativa (88%). This project intends to analyze the function of two novel genes. Semi-quantitative RT–PCR, qRT–PCR, GUS histochemical, and Promoter cloning were used to determine the expression and localization of the new genes. The expression and subcellular localization of LcFIN2 and LcFIN3 were detected by confocal laser scan microscope system and immunoblot analysis. The possible molecular mechanism of the novel genes were studied by using co-immunoprecipitation, yeast two hybrid, electrophoretic mobility shift assay. Furthermore, the phenotype, physiological and biochemical changes under freezing stress treatment of transgenic plants will be analyzed. With those work, the function and molecular mechanisms of two novel genes resistant to freezing will be confirmed. The implementation of this project will provide excellent gene resources for genetic improvement in forage and wheat.

羊草是我国重要的禾本科牧草，抗逆性强，能够在极端低温- 47.5℃条件下生存。挖掘羊草优异抗逆基因资源，对理解羊草抗逆分子机制具有重要意义。本人前期克隆并研究了两个羊草极端低温诱导新基因LcFIN2和LcFIN3，两个基因编码的蛋白均为未知功能蛋白且定位于叶绿体；LcFIN2与其它物种同源性较低，LcFIN3与水稻未知蛋白同源性为88%。本项目拟在此基础上开展下列研究：利用半定量及定量PCR、启动子克隆及GUS组织化学定位等确定新基因的表达定位；采用共聚焦激光扫描显微镜及免疫印迹技术进行新基因编码蛋白的亚细胞定位；利用免疫共沉淀、酵母双杂交、凝胶阻滞电泳迁移等实验研究新基因可能的分子作用机制；用转基因植物研究新基因抗冻的生物学功能等。目标是获得新的、具有明显抗冻功能的基因，阐明其可能的表达调控通路，为我国牧草及小麦等作物抗冻分子改良提供有价值的基因资源。

羊草（Leymus chinensis，Trin.）Tzvel）是欧亚草原东部广泛分布的一种重要牧草，对极端低温具有较高的耐受性。挖掘羊草优异抗逆基因资源，对理解羊草抗逆分子机制具有重要意义。在本研究中，我们明确了一个新的羊草冷冻诱导叶绿体编码蛋白LcFIN2的功能。结果表明，LcFIN2基因受低温、NaCl和ABA胁迫诱导表达。过表达LcFIN2提高了转基因拟南芥在低温胁迫下的存活率。同时，水稻中的异源表达LcFIN2，不仅可以提高转基因水稻在冷胁迫条件下的存活率，降低低温对水稻的膜损伤，而且在低温胁迫下，转基因水稻的叶绿素含量、光系统II的量子光化学效率 (ФPSII)、非光化学猝灭（NPQ）、净光合速率（Pn）、叶绿体核糖体相关基因和光合相关基因的表达均较高。此外，羊草LcFIN3基因的（重命名为LcSAIN3）初步研究结果表明，该基因可以提高转基因植物的抗盐性。通过本研究得到具有明确功能的羊草抗性新基因，可用于水稻等作物的耐低温及抗盐分子改良，对于作物遗传改良具有积极的意义。