【作者】 秦鑫；

【导师】 李勃； 潘建伟；

【作者基本信息】 兰州大学 ， 生物学·细胞生物学， 2022， 硕士

【摘要】 CBLs-CIPKs信号通路被认为是调控植物抗盐性的关键信号转导机制之一。水稻是世界重要的粮食作物,其CBLs-CIPKs在耐盐性中的功能还有待深入探索,因此研究水稻CBLs-CIPKs信号通路在盐胁迫信号转导中的调控机制对培育抗盐水稻品种具有重要意义。本文运用细胞生物学和分子遗传学方法,初步研究了OsCBL2/3-OsCIPK17复合体在调节拟南芥盐胁迫适应性中的分子机制。本论文将OsCBL2、OsCBL3和OsCIPK17基因分别在拟南芥中过表达并获得稳定的转基因株系。实验结果揭示了OsCBL2/3与OsCIPK17相互作用并将其从细胞质中招募到液泡膜上参与盐胁迫信号转导的生物学过程。本论文主要的研究结果如下:1.OsCIPK17在0.5μM ABA、150 m M Na Cl和300 m M甘露醇模拟的渗透胁迫处理下被显著诱导表达上调。OsCIPK17拟南芥过表达株系萌发率在逆境条件下(ABA、Na Cl和渗透胁迫)显著提高,这表明OsCIPK17在种子萌发阶段的抗逆性中发挥了重要作用,该机制可能与ABA信号通路有关。2.150 m M Na Cl处理下,拟南芥过表达OsCIPK17株系较野生型主根伸长显著提高,且该突变表型随着培养基Ca2+浓度的降低(小于0.5 m M)而消失,说明OsCIPK17参与的盐胁迫信号转导受到Ca2+的严格调控。3.酵母双杂交和双分子荧光互补实验证明OsCIPK17与OsCBL1、2、3、4、5和7在体外存在物理的相互作用。根据同源性分析和文献查阅选取OsCBL2和OsCBL3做进一步研究,研究结果发现ABA、Na Cl和渗透胁迫处理后同样可以显著诱导其表达上调,这一结果与OsCIPK17的表达情况相似,表明这三个基因协同参与胁迫信号的转导。4.过表达OsCBL2和OsCBL3到拟南芥同样发现盐胁迫条件下转基因株系较野生型主根伸长显著增加,这一表型与OsCIPK17的转基因株系相似。5.将OsCBL2-RFP和OsCBL3-RFP拟南芥株系分别与拟南芥液泡膜标记株系(VHA-a3-GFP)杂交。激光共聚焦显微镜下观察发现OsCBL2和OsCBL3与VHA-a3-GFP的信号重叠,这表明OsCBL2和OsCBL3定位于液泡膜。6.将拟南芥液泡膜标记株系(SYP22-RFP)与拟南芥OsCIPK17-GFP转基因株系杂交,发现正常情况下OsCIPK17定位于胞质,而在盐胁迫处理下在液泡膜积累。将拟南芥OsCBL2-RFP和OsCBL3-RFP株系分别与拟南芥OsCIPK17-GFP株系杂交,发现OsCBL2和OsCBL3分别与OsCIPK17共定位于液泡膜,表明OsCBL2和OsCBL3可能招募细胞质中的OsCIPK17到液泡膜行使调控功能。以上的实验证明OsCBL2/3-OsCIPK17复合体具有响应盐胁迫正调控主根伸长乃至植物抗盐性的功能。OsCIPK17在盐胁迫下被OsCBL2/3招募到液泡膜,推测该复合体可能在液泡膜通过调控物质(Na+、H+或其他抗逆相关的分子)跨膜转运参与调控植物对盐胁迫的适应性。上述结果在异源表达系统获得,下一步需在水稻中逐一验证。更多还原

【Abstract】 CBLs-CIPKs signaling pathways are considered to be one of the key signal transduction mechanisms that regulate salinity resistance in plants.Rice is an important food crop in the world,and the function of CBLs-CIPKs in salt tolerance needs to be fully characterized.Therefor it is of great significance for rice yield and quality to study the role of rice CBLs-CIPKs signaling pathway in response to salt stress.In this study,using cell biology and molecular genetics methods,the molecular mechanism of the OsCBL2/3-OsCIPK17 complex in regulating plant salt tolerance was studied in Arabidopsis.We overexpressed OsCBL2,OsCBL3 and OsCIPK17 in Arabidopsis individually to obtain stable transgenic strains.Experiments revealed that OsCBL2/3 interacts with OsCIPK17 and recruits OsCIPK17 from the cytoplasm onto vacuolar membranes to participate in salt stress signal transduction.The main findings of this study are as follows:1.OsCIPK17 is up regulated by 0.5 μM ABA,150 m M Na Cl and 300 m M Mannitol simulates osmotic stress in rice.The transgenic Arabidopsis lines overexpressing OsCIPK17 displayed significantly enhanced germination rate under treatments such as ABA,Na Cl and osmotic stress,indicating that OsCIPK17 plays an important role in stress tolerance at the seed germination stage,which may be related by ABA signaling pathway.2.When treated these transgenic plants lines with 150 m M Na Cl,significant elevation in primary root growth relative to wild type was observed under normal condition.Furthermore,when external of Ca2+ concentration was lower 0.5 m M,such mutative phenotype was absent.These results suggested that OsCIPK17 is involved in regulating primary root elongation in response to salt stress which is under control of Ca2+ signals.3.We investigated the possible interaction between the OsCIPK17 and 10 OsCBLs by both Yeast Two-Hybrid(Y2H)and Bimolecular Fluorescence Complementation(Bi FC).The result revealed that OsCIPK17 interacts with OsCBL1,2,3,4,5 and 7 physically in vitro.Therefore,OsCBL2 and OsCBL3 were selected for further analysis.The expression levels of OsCBL2/3 after treatments of ABA,Na Cl and Mannitol were significantly increased compared to controls,indicating that these 3 genes were involved in the transduction of these stress signals.4.The primary root elongation of transgenic Arabidopsis lines overexpressing OsCBL2 and OsCBL3 were significantly increased relative to the wild type after Na Cl treatment,which was similar to that of the transgenic lines overexpressing OsCIPK17.5.The Arabidopsis transgenic line overexpressing OsCBL2-RFP and OsCBL3-RFP were crossed with Arabidopsis tonoplast marker line(VHA-a3-GFP),respectively.Signals of OsCBL2/3 and VHA-a3-GFP were found to be overlapping under laser confocal microscopy,indicating OsCBL2 and OsCBL3 were localized on the tonoplast.6.On the other hand,SYP22-RFP,a Arabidopsis vacuolar membrane marker line,was crossed with Arabidopsis line overexpressing OsCIPK17.Interestingly,OsCIPK17 was shown localized in the cytoplasm under normal conditions,but accumulated on the tonoplast after salt stress.Furthermore,OsCBL2-RFP and OsCBL3-RFP lines were crossed with Arabidopsis overexpressing OsCIPK17-GFP.OsCBL2 and OsCBL3 were found to co-locate on the tonoplast with OsCIPK17.These results suggest that OsCBL2 and OsCBL3 may recruit OsCIPK17 from the cytoplasm to the tonoplast to perform regulatory functions.Taken together,these results suggest that OsCBL2/3-OsCIPK17 complex could positively regulate root elongation in response to salt stress.OsCIPK17 is likely recruited onto the tonoplast by OsCBL2/3 under salt stress.It is speculated that this complex may be involved in regulating plant salt tolerance by regulating the transmembrane transport of substances(Na+/H+ or other stress-related molecules)across the tonoplast.The above results are obtained in the heterologous expression system,and the next step needs to be verified in rice.更多还原