【作者】 周述波；

【导师】 肖浪涛；

【作者基本信息】 湖南农业大学 ， 植物学， 2023， 博士

【摘要】 海南山兰稻(Oryza sativa L.var Shanlan)又名“山栏稻”,是海南黎苗族等少数民族筛选保存下来的一类特有旱稻资源,抗旱性强,从山兰稻中挖掘抗旱优异基因具有重要的理论意义和应用价值。目前,山兰稻的抗旱生理特性缺乏系统研究,相关抗旱基因及其分子机制尚未见相关报道。因此,本研究以18个山兰稻品系为材料,筛选出抗旱水平差异显著的两个品系,结合生理生化、分子生物学、生物信息学、转录组学和基因编辑等实验手段,系统分析了山兰稻的生理特性与抗旱分子机制,主要研究结果如下:1.筛选出不同抗旱能力的代表性品系并比较了其抗旱表型及生理特性差异用25%的聚乙二醇6000(Macrogol 6000,PEG6000)模拟干旱处理18种山兰稻幼苗,发现山兰稻1号(SLD1)复水效果较差,山兰稻10号(SLD10)复水效果较好,两品系抗旱水平差异显著;干旱胁迫下,SLD1气孔密度大于SLD10,SLD1净光合效率比SLD10降低幅度更显著,SLD1气孔导度与蒸腾速率显著升高,水分利用效率降低;SLD1中过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)的酶活性均低于SLD10。2.通过多组学联合分析挖掘到抗旱的候选基因ZOS7转录组分析发现,正常水分条件下,两个品系间存在差异表达基因2243个;相比正常水分条件,干旱胁迫下SLD10上调基因243个,下调基因188个。结合靶向代谢组学开展多组学联合分析,在SLD10中发现412个高表达的干旱响应基因,其中C2H2型锌指蛋白基因ZOS7(Os07g0593000)与抗旱性密切相关。3.解析了ZOS7介导的抗旱分子机制zos7缺失突变体的叶片气孔密度增加,表面蜡质沉积减少,水分散失严重;在干旱胁迫下突变体植株发育延缓,分蘖数、生物量和产量降低,光合效率及抗氧化酶活性降低。ZOS7过表达植株叶片气孔密度降低,光合效率与生物量有所提高,ZOS7基因对水稻抗旱具有正向调控作用。干旱胁迫下,ZOS7基因受脱落酸(abscisic acid,ABA)诱导表达,其在植株体内表达量上升。进一步研究发现ZOS7和MYB60蛋白间存在互作,可形成复合体,调控蜡质合成相关基因GL1-8,PDR8和CER1等表达,尤其可通过识别CER1基因启动子,正向调控CER1基因表达从而促进抗旱。此外,ZOS7基因抑制气孔密度基因EFP9表达,使过表达植株中气孔密度降低,从而减少水分散失、增加抗旱。山兰稻具有较为复杂的干旱响应机制,其中干旱和ABA可诱导ZOS7基因显著上调,并且在抗旱性强的山兰稻中高度表达。通过基因敲除和过表达证明ZOS7基因正向调控水稻抗旱反应,ZOS7与MYB60可共同识别CER1基因的上游顺式作用元件以激活蜡质合成相关基因CER1的表达。ZOS7基因在体内表达上调可降低气孔密度以增强抗旱性。本研究从山兰稻中挖掘到了抗旱关键基因ZOS7并初步解析了ZOS7介导的水稻抗旱机理,为水稻抗旱研究提供了理论参考。更多还原

【Abstract】 Hainan Shanlan upland rice(Oryza sativa L.var Shanlan),alternatively known as "Shan Lan Rice",is an important upland rice crop resource preserved by the Li and Miao ethnic minority in Hainan province.It has strong drought tolerance.Mining excellent genes for drought tolerance has important theoretical significance and application value.At present,there is a lack of systematic research on the drought-tolerant physiological characteristics of Shanlan upland rice,and there are no related reports on the drought-tolerant genes and their molecular mechanisms.Therefore,this study used 18 Shanlan upland rice lines(SLD1-18)as materials,screened out two lines with significant differences in drought tolerance,combined with experimental methods such as physiology and biochemistry,molecular biology,bioinformatics,transcriptomics,and gene editing,to systematically analyze the physiological characteristics and drought tolerance molecular mechanism of Shanlan unpland rice.The main research results are as follows:1.Representative lines with different drought tolerance were screened out and their drought tolerance phenotypes and physiological characteristics were compared.Using 25% polyethylene glycol 6000(Macrogol 6000,PEG6000)to simulate drought to treat 18 kinds of Shanlan unpland rice seedlings,it was found that the rehydration effect of SLD1 was weaken,and that of SLD10 was rehydrated.The drought tolerance level of the two lines was significantly different.Under drought stress,the stomatal density of SLD1 was greater than that of SLD10,the net photosynthetic efficiency of SLD1 decreased more significantly than that of SLD10,the stomatal conductance and transpiration rate of SLD1 increased significantly,and the water use efficiency decreased accordingly.The enzyme activities of catalase(CAT)and superoxide dismutase(SOD)were lower than those of SLD10.2.A candidate gene ZOS7 for drought tolerance was mined through joint multi-omics analysis.Transcriptome analysis suggests that under normal water conditions,there were 2243 differentially expressed genes between the two lines.Compare with normal water conditions,there were up-regulated 243 genes and down-regulated 188 genes under drought stress in SLD10 relative to SLD1.Combining multi-omics analysis with targeted metabolomics,412 highly expressed drought-responsive genes were found in SLD10,among which the C2H2-type zinc finger protein gene ZOS7(Os07g0593000)was closely related to drought tolerance.3.The molecular mechanism of ZOS7-mediated drought tolerance was analyzed.The leaf stomatal density of zos7 mutants was increased,waxy deposition on the surface was reduced,and water loss was severe,compared to ZH11;Under drought stress,the mutant plants had delayed plant development,decreased tiller number,biomass and yield,and decreased photosynthetic efficiency and antioxidant enzyme activities.The stomatal density of ZOS7 overexpressed plants decreased,and the photosynthetic efficiency and biomass boosted compared to ZH11.The ZOS7 gene had a positive regulatory effect on drought tolerance of rice.Under drought stress,the expression of ZOS7 gene was induced by abscisic acid(ABA).Further studies had found that there was an interaction between ZOS7 and MYB60 proteins,which could form a complex and regulate the expression of wax synthesis-related genes GL1-8,PDR8 and CER1,especially by recognizing the CER1 gene promoter,resulting in positively regulating CER1 gene expression to promote drought tolerance.In addition,the ZOS7 gene inhibits the expression of the stomatal density gene EFP9,which reduced the stomatal density in overexpressed plants,thereby reducing water loss and increasing drought tolerance.Shanlan upland rice had a complex drought response mechanism,in which drought and ABA could induce a significant up-regulation of ZOS7 gene,and it was highly expressed in that Shanlan upland rice lines with strong drought tolerance.Knockout and overexpression experiments proved that the ZOS7 gene positively regulated the rice drought response,ZOS7 and MYB60 could jointly recognize the upstream cis-acting element of the CER1 gene to activate the expression of the wax synthesis-related gene CER1.Up-regulation of ZOS7 gene in vivo could reduce stomatal density to enhance drought tolerance.In this study,the key gene ZOS7 for drought tolerance was excavated from Shanlan upland rice,and the drought tolerance mechanism mediated by ZOS7 was preliminarily analyzed,which provided a theoretical reference for the study of drought tolerance in rice.更多还原