钙离子介导的拟南芥感受盐胁迫相关基因SICI1的功能鉴定

Salinization is a major factor limiting the productivities of agricultural production. How the plants sense the salt signal is a longstanding question. High salt triggers Ca2+ influx in plant cells, leading to a transient increase in [Ca2+]i. This is believed to be involved in salt sensing. However, the underlying molecular mechanism is unknown. In the previous study, we have screened several mutant candidates which failed to exhibit this early [Ca2+]i elevation upon NaCL stimulus, and isolated a calcium signal reduced gene (Salt-induced [Ca2+]i increases 1 gene, SICI1) by high-throughput Arabidopsis mutant screening system based on aequorin and Ca2+ imaging. SICI1 protein has 6 transmembrane domains by bioinformatic analysis. We will analyse whether SICI1 have ion channel characterization in HEK293 cell and in plants by electrophysiology and patch clamp. The subcellular localization and tissue-specific expression pattern were detected by constructing different vectors. Some physiological parameters related to the salt stress were assessed in mutants and over-expressed transgenic plants. In a word, We will integrate genetic, cellular and molecular approaches with calcium image and electrophysiological technologies to explore the mechanistic issues of signal transduction during plant response to salt stress. Discoveries from this research will provide knowledge-base, genetic resources including novel molecular markers for the breeding of salt tolerant crops.

随着全球环境变化，土壤盐渍化面积不断扩大，盐胁迫成为限制农作物生长发育的重要环境因子。植物如何感受盐胁迫信号成为人们长期关注又亟需解决的问题。盐胁迫引起胞内游离钙离子浓度([Ca2+]i)迅速增加，但内在分子机制尚未明确。利用本实验室建立的一整套基于水母发光蛋白的钙离子成像技术、高通量筛选盐胁迫诱导下[Ca2+]i升高不显著的缺陷型插入突变体技术体系，分离克隆到盐胁迫诱导下钙信号减弱的基因SICI1，其编码蛋白存在6个跨膜结构域。本项目拟以此为基础，构建GUS/GFP载体检测SICI1基因的表达模式及亚细胞定位、膜片钳技术分析其蛋白离子通道特性、qPCR检测下游抗/耐盐基因的表达，构建超表达株系进行盐胁迫表型及相关生理指标的测定，RNA-seq研究sici1中盐胁迫信号转导途径，阐明SICI1基因在Ca2+介导的拟南芥抗/耐盐过程中的作用。该研究对于抗/耐盐农作物的培育具有重要指导意义。

盐胁迫是影响植物生长、发育以及产量的重要环境因子，同时又是一个较为复杂的非生物胁迫因子。植物如何感受盐胁迫信号成为人们长期关注又亟需解决的问题。盐胁迫引起胞内游离钙离子浓度([Ca2+]i)迅速增加，但内在分子机制尚未明确。本项目通过筛选拟南芥中在盐胁迫条件下钙信号响应缺失突变体sici1，确定SICI1基因在盐胁迫感应中的作用。结果表明sici1 突变体对盐刺激具有专一性，SICI1基因定位于细胞内膜上，编码蛋白含有7个跨膜结构域。sici1突变株更不抗盐，不抗旱，但在低钙高钙胁迫下基本无表型。K+ transporter基因缺陷体 E. coli LB650实验及Fura-2实验结果表明SICI1蛋白可能无离子通道功能，但有可能是离子通道相关的互作蛋白，作用机理需要进行进一步研究。此外，我们发现并分析了盐胁迫与低温胁迫之间所产生钙信号既相互独立，又有相关性。该研究的进行对抗/耐盐农作物的培育提供了重要指导意义。