高羊茅耐高温生理分析及HSP17.8和HSP17.9分子克隆

发布时间：2017-12-31 21:09

本文关键词：高羊茅耐高温生理分析及HSP17.8和HSP17.9分子克隆　出处：《中国科学院武汉植物园》2017年硕士论文　论文类型：学位论文

【摘要】：高羊茅(Festuca arundinaacea Schreb.)是一种重要的冷季型草坪草,普遍应用在全球温带和过渡带地区,但夏季高温使高羊茅草坪质量下降,严重时引起大面积死亡。因此,高温被认为是发展和利用高羊茅的主要限制因子。热锻炼诱导小分子热激蛋白(sHSP)的积累与植物的耐热性密切相关,因此,研究小分子热激蛋白应答高温胁迫的机制对于高羊茅的遗传育种具有重要的理论意义和应用价值。本研究以高羊茅热敏感'PI234881'、耐热'PI578718'种质为材料,研究高温以及干旱胁迫对高羊茅生理特性的影响,挖掘高羊茅优异耐热基因,揭示转录记忆效应的基因FaHSP77.8和FaHSP17.9的分子特征,为深入研究高羊茅耐热基因功能提供分子基础。主要研究结果如下:1.高羊茅应答高温干旱胁迫的生理特性高温以及干旱胁迫显著提高热敏感和耐热型高羊茅叶片丙二醛(MDA)含量和电解质渗透率(EL),导致细胞膜稳定性下降,且在不同品种中存在较大变异。热敏感型高羊茅超氧化物歧化酶(SOD)活性降低更为显著,H202含量增加更多,表明其受胁迫程度更严重。高羊茅通过提高SOD、过氧化物酶(POD)及过氧化氢酶(CAT)等酶的活性来保护自身免受伤害。高温以及干旱胁迫显著降低高羊茅叶片叶绿素含量,通过快速叶绿素荧光动力学曲线分析发现,双重胁迫对高羊茅光合性能指数PIABs、推动力DFABs和最大光化学效率(pPo的影响最大,数值最低,高羊茅品种在逆境胁迫下光合特性都有所下降,热敏感型高羊茅光系统Ⅱ功能受损更为严重。2.高羊茅小分子热激蛋白基因的表达特性通过qRT-PCR分析高羊茅小分子热激蛋白HSP17.8、HSPI7.9、HSP18.6和HSP21.9在34℃(4h)-22℃(20h)次高温循环胁迫模式下的表达水平。结果表明,经过多次高温循环胁迫,HSP17.8和HSP17.9表达量显著高于第一次高温胁迫,即具有转录记忆效应;HSP18.和HSP21.9的表达量在每次热胁迫时趋于相近水平,甚至有下降的趋势,为'非记忆基因'。3.高羊茅FaHSP17.8和FHSP17.9的分子克隆基于高羊茅高温胁迫转录组数据,克隆高羊茅FaHSP17.8和HaHSP17.9,cDNA编码区序列全长分别为 489 bp、459bp,分子量分别为17.8K,17KDa,基因组gDNA均无内含子序列;氨基酸多重序列比对和进化树构建表明,FaHSP17.8和FaHSP17.9分别和小麦HSP17.8、水稻HSP17.9有很高的序列同源性(94%,91%),它们的亲缘进化关系在不同分支上,分别属于细胞质Ⅱ类和Ⅰ类小分子热激蛋白;亚细胞定位显示,FaHSP17.8和FaHSP17.9均定位在细胞质/细胞核中;组织特异性表达结果发现,FaHSP17.8和FaHSP17.9在叶片中表达量最高,其次是茎,根中最少。4.高羊茅小分子热激蛋白FaHSP17.9启动子克隆利用染色体步移技术,克隆FaHSP17.9启动子序列,并对其ATG上游917 bp序列进行初步分析。通过PlantCARE数据库,在线预测该基因的顺式作用元件,发现高温胁迫应答元件HSE-motif(CCAAT),可能对FaHSP17.9的热诱导性能起到主要作用。综上所述,我们研究高羊茅抗氧化酶代谢系统的响应和PSⅡ对高温及干旱适应性机制,挖掘高羊茅耐热记忆基因,分析其基本表达特性。本研究为进一步研究记忆基因FaHSP17.8和FaHSP17.9表观遗传修饰模式和基因功能,进而培育耐热高羊茅新品种提供重要的分子基础。
[Abstract]:Tall fescue (Festuca arundinaacea Schreb.) is an important cool season turfgrass, widely used in the global temperate zone and transitional zone, but the summer high temperature makes the turf quality of tall fescue decline caused by severe death in a large area. Therefore, high temperature is considered to be the main factor limiting the development and utilization of tall fescue. Exercise induced heat small heat shock proteins (sHSP) heat accumulation and plant are closely related, therefore, research on the mechanism of small heat shock proteins in response to high temperature stress has important theoretical significance and application value for genetic breeding of tall fescue. The tall fescue heat sensitive'PI234881','PI578718' resistant germplasm materials of high temperature and effects of drought stress on physiological characteristics of tall fescue, tall fescue mining excellent heat resistant gene, molecular characteristics reveal transcriptional memory effect of gene FaHSP77.8 and FaHSP17.9, to study To provide the molecular basis function of fescue thermotolerance gene. The main results are as follows: 1. tall fescue in response to high drought stress physiological characteristics of high temperature and drought stress increased significantly in tall fescue leaves MDA heat sensitive and heat tolerant (MDA) content and electrolyte permeability (EL), leading to a decrease in cell membrane stability, and there is a large variation in different varieties. Thermosensitive tall fescue superoxide dismutase (SOD) activity decreased significantly, the content of H202 increased more, showed that the extent of stress is more serious. Tall fescue by increasing SOD, peroxidase (POD) and catalase (CAT) activity to protect themselves from harm of high temperature and drought stress significantly. Reduce the chlorophyll content, the fast chlorophyll fluorescence kinetics analysis found that stress on tall fescue photosynthetic performance index of PIABs, DFABs and the maximal photochemical driving force Efficiency (pPo impact, the lowest value, varieties of Tall Fescue under stress photosynthesis decreased, thermosensitive tall fescue PSII function is damaged more serious expression characteristics of tall fescue.2. small heat shock protein gene by qRT-PCR analysis of tall fescue small molecular heat shock protein HSP17.8, HSPI7.9. HSP18.6 and HSP21.9 at 34 DEG C (4h) -22 (20h) high temperature cyclic stress levels under the model. The results show that after high temperature cyclic stress, the expression of HSP17.8 and HSP17.9 was significantly higher than the first temperature stress, which has transcriptional memory effect; expression of HSP18. and HSP21.9 to levels similar to those in each heat stress even when there is a downward trend, as' molecular memory gene'.3. in tall fescue FaHSP17.8 and FHSP17.9 cloning of transcriptome data based on high temperature stress of tall fescue, tall fescue and HaHSP17.9 FaHSP17.8 clone, cDNA encoding region The sequence length was 489 BP, 459bp, molecular weight of 17.8K and 17KDa respectively, there were no genomic gDNA intron sequences of amino acids; multiple sequence alignment and phylogenetic tree showed that FaHSP17.8 and FaHSP17.9 respectively, and HSP17.8 of wheat and rice HSP17.9 had high sequence homology (94%, 91%), their evolutionary relationship in different branches, belong to the cytoplasm of class II and class I small heat shock protein; subcellular localization showed that FaHSP17.8 and FaHSP17.9 were localized in the cytoplasm / nucleus; tissue specific expression of FaHSP17.8 and FaHSP17.9 showed the highest expression level in leaves, followed by stems, roots of tall fescue at least.4. small heat shock protein FaHSP17.9 promoter was cloned by chromosome walking technology, cloning of FaHSP17.9 promoter sequence, and the ATG 917 bp upstream sequence was analyzed. By PlantCARE database, online prediction of the gene The cis acting elements, found that high temperature stress response element HSE-motif (CCAAT), may play a major role in inducing properties of FaHSP17.9 heat. In summary, we studied the response of tall fescue antioxidant metabolism system and PS II to high temperature and drought adaptation mechanism, mining of tall fescue heat-resistant memory gene, analysis of the basic characteristics of this expression. For the further study on the memory of FaHSP17.8 gene and epigenetic modifications and gene function FaHSP17.9, and tolerant tall fescue provide important molecular basis.

【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S688.4

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