棉花GhSAG基因的功能分析和分子机理研究

Cotton is an important economic crop. Its growth and yield are severely inhibited by high salinity and drought, especially at the germination and emergence stages.So the studies for discovering the mechanisms that control the germination and emergence of cotton seeds under abiotic stresses can provide the theoretical basis for cotton molecular breeding. Phytohormone ABA plays important roles in response to abiotic stresses, such as drought and salt. ABA signaling is regulated by many protein complexes. Studies have revealed that AAA+ domain containing protiens play important roles in the assembling, transporting and regulating of these protein complexes. For the first time,our results have displayed that AtSAG,an AAA+ domain containing protien from Arabipsis, participated in the regulation of ABA signaling via an ABI3-ABI5 dependent pathway. However, the function of AAA+ domain containing protien from cotton has not been characterized.The molecular mechanism of SAG in the the relationship with ABI5 and other ABA signal components is also very limitid. So we will investigate the function of GhSAG from cotton by overexpression of it in Arabidopsis sag mutant and cotton, identify proteins interacting with SAG, genes downstream of it and phosphorylated protein kinases of it. We will investigated the function of AAA+ domain of SAG by amino acid mutantion, and characterize the changes of seed storage proteins, oil bodies and lipid during cotton seed germination and emgergence. The purposes of this study are to clarify the function of GhSAG, to clarify the signaling pathway and molecular mechanism controled by SAG, to clarify the function of AAA+ domain in SAG, and to provide valuable evindence of SAG for its potential ability for breeding abiotic resistant crops.

盐碱和干旱等条件严重影响棉花的出苗、保苗和壮苗。因此研究棉花种子萌发出苗期对盐和干旱等胁迫响应的生理及分子机理具有重要意义。脱落酸（ABA）在干旱、盐等胁迫响应中起主要作用,受到多种蛋白复合体的调控。AAA+蛋白在蛋白复合体组装、转运及活性调节等方面扮演重要角色。我们首次报道了一个AAA+蛋白AtSAG参与了拟南芥萌发时的ABA信号调节。但棉花GhSAG的功能，SAG调节ABA信号的作用机理还不清楚。因此本项目将通过棉花GhSAG过表达株系的表型鉴定，互作蛋白、下游基因和磷酸化蛋白激酶的分离鉴定，AAA+结构域系列突变载体对sag突变体表型的互补分析，及种子贮藏蛋白和油体、脂类在胁迫条件下棉花种子萌发出苗期的变化等，阐明SAG调控的ABA信号途径和作用的生理及分子机制，为作物抗逆育种提供新的理论基础。

盐和干旱等逆境是植物产量和品质的主要限制因子，研究植物对盐和干旱等逆境响应的生理及分子机理具有重要意义。本项目首次报道了一个AAA+蛋白SAG参与植物萌发时的ABA信号调节。通过对棉花GhSAG在野生型和sag突变体中过量表达株系的分子和表型鉴定，发现GhSAG过量表达株系在萌发期对盐胁迫、渗透胁迫和ABA敏感。这与拟南芥sag突变体表型一致而与AtSAG过量表达株系表型相反，说明GhSAG是AtSAG的突变体。与拟南芥SAG氨基酸序列比较发现棉花SAG在C端有大片段缺失，包括Tudor蛋白与蛋白互作结构域。sag突变体种子大小和发育相关基因及油体检测结果表明SAG通过调控油体和贮藏蛋白基因表达而调控种子发育和萌发。CHOP-PCR和甲基化敏感位点测序结果表明SAG影响DNA甲基化。该过程依赖ROS1。全基因组甲基化水平检测结果表明SAG仅影响10%的DNA甲基化。通过AtSAG过量表达株系和野生型转录组分析和下游基因基因功能鉴定，发现SAG通过负调控GhPLATZ1的表达调控植物的耐盐性和ABA响应；通过正调控GhTPS11的表达调控低温条件下的种子萌发。说明超表达拟南芥SAG 显著提高转基因植物萌发期对盐和干旱胁迫的抗性，在植物抗逆育种中具有一定的应用价值。