【作者】 于二汝；

【导师】 周永明；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 高温是影响农业生产的非生物胁迫因子之一。长江流域是我国油菜的主产区,该地区在种子灌浆成熟期经常遭遇短期或连续30度以上的高温,造成种子高温逼熟,严重影响种子产量和含油量。但目前还缺乏对油菜耐高温逼熟的遗传和分子基础的了解。模式植物拟南芥中热激转录因子研究已取得重要进展,但如何有效应用到作物遗传改良还有待探索。针对这些问题,本研究主要开展了三个方面的工作：1)以开花20d后的油菜果皮和种子为材料,利用油菜95K表达序列芯片分析两种组织在热处理后24h和48h基因表达变化的情况,为研究油菜抗热反应提供更广泛的基因源。2)利用拟南芥热激转录因子多突变体,从表型、表达调控和蛋白互作分析以及遗传调控途径等方面研究HsfA与HsfB两个亚家族基因的功能和潜在的调控关系。3)根据油菜热激表达谱结果,以受热胁迫诱导表达的拟南芥基因H38(AT4G23493)为分析对象,从基因表达、亚细胞定位、生理生化反应以及生长发育调节等方面研究了其功能。主要研究结论如下：1.在热处理后的油菜芯片中共检测到1248个上调和898个下调表达的基因,其中在果皮中检测到925个上调和581个下调表达基因,在种子中共检测到837个上调和383个下调表达基因。在果皮和种子中,这些转录水平的变化可能为揭示油菜角果期抗热的分子机制提供线索。2.上调基因中有40.9%(511)同时存在于种子和果皮中,然而下调表达的基因中只有66个同时存在于两个组织中,并且上调基因的平均变化倍数(2.1-72.8)比下调(2.0-5.6)的更加剧烈。在共同诱导表达的基因中,与胁迫相关的基因以及与RNA和蛋白质相关等基因显著富集,其中包括热胁迫反应的标志性基因如Hsfs、 Hsps、DREB2a、ROF2、GolSl和MBF1c等,以及CYP707A4和细胞色素bd泛醌氧化酶等以前未鉴定到的基因。3.在果皮中共检测到411个上调和514个下调表达基因,而这些基因在种子中的表达没有受到影响。其中,最突出的是21个参与硫苷代谢的基因被同时下调表达,另外一些表达改变的基因则参与光合作用,水、糖分以及离子转运等几个主要代谢途径,这些基因表达量的变化可能与热处理后油菜种子产量以及品质直接相关。4.在种子中检测到325个上调和314个下调表达基因,其在果皮中的表达没有明显变化。与果皮类似,我们共检测到12个花青素合成途径上的基因表达受到抑制,此外与种子储藏蛋白、植物激素、油脂代谢等重要代谢途径相关的基因的表达也发生变化。5.在所有的差异表达基因中,有1/3编码功能未知蛋白(果皮中有484个,种子中有398个),其中502个在拟南芥中能找到同源序列,181个则为油菜特有基因。因此,在植物中可能还存在许多与抗热相关的功能未知基因以及物种特有基因。6.根据芯片数据,我们挑选了8个基因并获得了相应的拟南芥突变体。在种子萌发、幼苗、成熟植株(开花之前)等多个时期对这些突变体进行热处理,发现h25、h29与h38至少在一种热处理条件下表现热敏感。7.通过对双突变体的分析发现,HsfA3位于HsfA2下游协同调节Hsp18.1和Hsp25.3-P等的表达。采用酵母双杂交的方法检测到HsfA2与HsfA3可以通过各自的OD结构域进行互作。利用HsfB的双突变体和三突变体,从抗热表型和靶标基因的表达变化等角度阐述了HsfB的功能及其之间的关系。8.在芯片中发现的热应答基因H38(AT4G23493),在植物界中保守的。在拟南芥中,H38主要在成熟的种子以及萌发过程中高丰度表达；其编码的蛋白定位于线粒体。启动子和基因表达分析发现H38在幼苗以及发育的角果中能显著被热诱导。h38突变体在苗期对热处理敏感,而在抽薹开花期热处理时能够显著积累花青素。9.采用生物信息学预测发现H38与GolS1、SK1、热激蛋白AT5G37670和AT2G19310等基因共表达,采用荧光定量的方法分析了共表达基因以及抗热的核心基因Hsf和Hsp和线粒体定位的抗热关键基因的表达变化,明确了H38的功能与调控网络。10.h38突变体的种子在萌发过程中对葡萄糖和ABA的耐受性显著增强,这种耐受性与渗透胁迫无关。此外,h38突变体的花器官、种子等显著比野生型增大,细胞学观察表明器官增大主要是由于细胞体积增大造成。更多还原

【Abstract】 High temperature stress is one of the major abiotic stresses in the world that severely restricts crop production. The Yangzi River Valley is the most important rapeseed oil production area in China, but this area usually suffers transitory or long period high temperature of over30℃at seed filling stage, which could result in heat-forced maturity, decreased oilseed yield and quality. However, the inheritance and molecular mechanisms of anti-heat-forced maturity in oilseed rape are still largely unknown. Heat resistance genes including heat stress transcription factors were well studied in model plant Arabidopsis, but how to apply the theoretical knowledge to genetically improve heat resistance of crops effectively is poorly understood. To address these issues, three major works were conducted in this study:1) Global transcription profiles in siliques of Brassica napus from20days after flowering after heat stress was analyzed using a95k Brassica60-mer GeneChip. The newly identified transcripts further enriched the reservoir of heat-responsive genes.2) Using double and triple mutants, functional analysis and potential regulation network of HsfAs and HsfBs were conducted in Arabidopsis by phenotypic analysis, gene expression and protein interaction.3) Functions of an unknown gene of H38(AT4G23493) that was discovered to be a potential heat regulator by microarray survey, were systematically studied from the aspects of gene expression, subcellular localization, physiological and biochemical studies and growth and developmental regulation analysis. The main conclusions of this study achieved are as follows:1. We detected1248up-regulated and898down-regulated genes in the heat stressed rapeseed siliques;925up-regulated and581down-regulated were detected in the silique walls (SW), and837up-regulated and383down-regulated genes in the seeds. The alteration of the transcripts may provide clues for revealing the mechanism for heat-resistance in the oilseed rape at the seed filling stage.2.40.9%(511) of the up-regulated genes are present in the seeds and silique walls simultaneously, however, only66genes were decreased in both organs; and the average fold-change of the up-regulated genes (2.1to72.8) was significantly larger than that of the down-regulated ones (2.0to5.6). Notably, stress-related genes and transcription factors were significantly enriched in both organs. Many of the up-regulated genes are known to be heat-marker genes, including13Hsfs and91Hsps, and DREB2a, ROF2, GolSl, MBFlc, and others such as CYP707A4and panthenol oxidized cytochrome bd enzyme which were not identified in heat response previously.3.411up-regulated and514down-regulated genes were detected in the silique walls, but not affected in seeds. Among them, some of the genes were involved in the major metabolic pathways of photosynthesis and transport (water, sugar and ion) and a cluster of21genes that are involving in glucosinolates metabolism were simultaneously reduced in heat stressed silique walls. All these altered expression may directly related to the decreased yield and quality of rapeseed after heat stress.4.325up-regulated and314down-regulated genes were altered in seeds and whose expression did not change significantly in the silique walls. Genes related to seed storage proteins, phytohormones, lip id metabolism and other important metabolic pathways were changed. Similar to the silique walls, we have detected as many as12genes that involved in the anthocyanin biosynthetic pathway were suppressed in heat stressed seeds.5.1/3of all the differentially expressed genes encodes protein with unknown functions (484in silique walls,398in seeds),502of which can be found their homologous sequences in Arabidopsis, while181were rapeseed specific genes. Therefore, there may be many unknown and species-specific genes that regulate plant thermotolerance.6. According to the microarray data,8genes were selected for further studies. Mutants of homozygous genes in Arabidopsis were obtained and confirmed by PCR and gene expression analysis. Further, thermotolerance assays were carried out at the stage of seed germination, young seedling and mature plants (before flowering) of each mutant; and h25, h29and h38were found to be heat-sensitive in at least one of the heat-treatment conditions. 7. From the phenotypic and expression analysis with the double mutants and single mutants of hsfA2and hsfA3under long-time recovery from heat stress showed that HsfA3was downstream of HsfA2and synergistically regulated the expression of HSP18.1and Hsp25.3-P. Moreover, HsfA2and HsfA3could strongly interact though their oligomerization domains by yeast two hybrid approach. Function analysis and regulatory study of three HsfBs were performed by phenotypic analysis after heat stress, and gene expression analysis of heat resistant symbol genes and potential target genes using double and triple mutants.8. H38was identified as a conserved gene with unknown function in the plant kingdom. In Arabidopsis, H38was mainly expressed in the mature and germination seeds and encodes the protein that was located in mitochondrial. H38was significantly induced after heat stress in seedlings and developing siliques by histochemical staining and qRT-PCR analysis, Mutant of H38was sensitive to heat stress at seedlings stage; while it accumulates more anthocyanins after heat stress at the early stage of flowering.9. Co-expression analysis revealed that expression of H38was intimately correlated to that of GolSl, SKI, heat shock protein AT5G37670and AT2G19310. The co-expression genes together with some representative Hsfs/Hsps and mitochondrial localized heat-related genes were studied by qRT-PCR, which provides important information of the regulatory network of H38.10. The tolerance of seed germination under glucose or/and ABA treatment was significantly enhanced in h38mutant; what is more the floral organs and seeds of h38were significant larger than the wild type which was mainly resulted from the increased cell size by cytology observations.更多还原