【作者】 周静；

【导师】 杨万年；

【作者基本信息】 华中师范大学 ， 植物学， 2016， 硕士

【摘要】 由高温而造成的热胁迫通常会打破细胞内稳态,对植物的生长发育产生不利影响。为了应对热胁迫,植物在长期的进化过程中已形成一系列适应机制。在植物体内由热激转录因子(Hsfs)调控的热激蛋白(Hsps)的积累被认为在热胁迫应答和耐热性方面有重要作用。虽然对Hsfs-Hsps应答通路已有一定了解,但该过程中的很多关键因子目前仍是未知的。为了找到调控热激基因表达的相关因子,我们建立了荧光素酶LUC(luciferase)筛选系统,即将Hsp18.2的启动子驱动LUC报告基因(AtHsp18.2pro:LUC)的构建转化到gl1拟南芥中,鉴定单拷贝、纯合的转基因植株(P-LUC)作为野生型材料,经EMS诱变,筛选热激处理后报告基因LUC表达升高或下降的突变体。利用该方法,我们获得了 LUC被激活的突变体hl307(highlight307),并对其进行研究。通过前期研究,我们已经知道突变体hl307在38℃处理1h后,与野生型相比其LUC的化学发光增强;通过图位克隆技术和二代测序,实验室已克隆到了突变基因,该基因的第2140个碱基由G突变为A,突变位点位于第六个内含子的5’端剪接位点,从而影响自身剪接;生物信息学分析显示该突变基因编码一种剪接因子,因此,我们把其命名为 SFH(Splicing Factor Relted t Heat Stress)。对突变体的进一步研究发现,突变体hl307在38℃处理0.5 h、1 h、2 h和5 h时,其LUC化学发光与野生型相比均显著性增强;突变体对高温敏感;在正常生长条件下,突变体hl307具有早花和植株矮小的表型;hl307与其等位突变系(T-DNA插入突变体)的杂交一代(F1)也表现出比野生型强的LUC发光,而hl307互补植株(Cmp)的LUC化学发光则介于野生型和突变体之间,说明上述表型的确是由SFH基因突变引起的;基因表达分析显示SFH基因在根、茎、叶、花和角果中都有表达,呈组成型表达。对部分Hsfs和Hsps的表达分析发现,大多数Hsfs和Hsps在突变体hl307中的表达水平都低于野生型P-LUC中的表达水平,为解释突变体高温敏感表型提供了依据。同时,SFH与GFP的融合蛋白的亚细胞定位显示SFH蛋白在细胞核中分布。综上所述:SFH蛋白对热激基因Hsp18.2表达起抑制作用,SFH蛋白与mRNA前体的剪切和热胁迫响应相关。目前的研究结果为进一步研究热激基因的表达调控机理以及阐明植物的耐热机制打下了基础。更多还原

【Abstract】 Heat stress caused by elevated temperature could disturb cellular homeostasis and lead to adverse impacts on plant growth and development.Plants have evolved a series of adaptive mechanisms to cope with heat stress.The accumulation of heat shock proteins(Hsps)regulated by heat stress transcription factors(Hsfs)is assumed to play a central role in the heat stress response and in thermotolerance in plants.We have a certain understanding of Hsfs-Hsps response pathway.However,many of the key factors mediating the heat response pathways remain unkown.In order to indentify regulators involed in heat stress,we established a forward genetic method for screening mutants with altered Hsp expressions.A firefly luciferase reporter gene driven by the Hsp18.2 was introduced into Arabidopsis thaliana glabrous1 mutants(Col-0 background).Seeds from a homozygous line harboring a single copy of Hsp18.2pro:LUC transgene was used as the wild type P-LUC,and mutagenized by EMS(ethyl methanesulfonate).Then the mutants with altered luminescence expression under heat stress treatment were screened by using a hypersensitive CCD camera.Through this method,we found a mutant named hl307 show elevated luciferase expression comparing with wild type and further studied.In our previous research,it was found that hl307 mutant displayed the elevated luciferase expression comparing with wild type under the treatment with 38℃ for 1 hour and the mutant gene was identified by map-based cloning and next-generation sequencing.In hl307,a nucleotide change of G2140A from the start codon was found in this mutant gene.The mutational site lies in the 5’ end splice site of the sixth intron,leading to the splicing change of this gene.The bioinformatics analysis showed that this gene encode a splicing factor.Therefor,the gene was designated SFH(Splicing Factor Related to Heat Stress).Our further study showed that the hl307 mutant displayed the obviously higher luciferase expression compared with the wild type under 38℃ treatment for half hour,1 hour,2 hours and 5 hours.In addition,hl307 is hypersensitive to heat stress.Under normal growth conditions,hl307 were early flowering and greatly reduced in size compared with P-LUC.T-DNA insertion lines of SFH gene identified from TAIR seed stocks were respectively crossed with hl307,the seedings of filial generation(F1)displayed the significantly elevated luciferase expression compared with WT.And luminescence of hl307 complementation transformants(Cmp)is between the luminescence of WT and hl307 mutant.Hence the above phenotypes observed in hl307 plants are indeed resulted from the mutation of SFH.SFH gene expresses in all tested tissues including roots,stems,leaves,flowers and siliques.Analysis of several Hsfs and Hsps expression showed that most Hsfs and Hsps expression level in hl307 mutants were lower than in P-LUC.This results provides some clues for explaining the phenotype that hl307 is hypersensitive to high temperature.Additionally,the image of the SFH-GFP fusion protein showed that SFH is localized in the nucleus.In conclusion,SFH protein can inhibit the expression of Hspl8.2 and may play important roles in pre-mRNA splicing and heat stress response.The current findings can help us to understand the mechanisms about thermotolerance in plants.更多还原