【作者】 蒋昌华；

【导师】 沈大棱； 明凤；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 月季（Rosa chinensis）为蔷薇属中能连续开花的观赏花卉植物,在园林绿化领域中享有极高的声誉,深受人们喜爱。由于全球“温室效应”现象日益明显,高温已成为制约植物生长和发育的主要环境因子,月季的生长发育也面临着高温逆境的严峻挑战,因此,如何提高月季对高温等逆境胁迫的抗性研究意义重大。高质量基因组DNA、总RNA的有效提取是月季分子生物学研究基础。月季富含单宁、多糖、酚等次生代谢物,严重影响其DNA、RNA的提取。本文通过对经典CTAB法的改进而获得高质量的月季DNA;筛选到植物RNAout试剂盒较适合月季总RNA提取;为了获得大量的实验材料,本文还建立了有效的月季快速繁殖体系。本文筛选到两个耐热性差异明显的月季品种:耐热的‘曼海姆宫殿’（SM）与不耐热的‘新十全’（KP）。38℃/3h热激后经双向电泳,获得SM在高温下差异表达的蛋白质点。经肽质谱分析,初步推定该蛋白质点与拟南芥小分子热激蛋白HSP17.5同源性高。同源克隆获得251bp的片段,根据序列设计3对反式PCR引物,以SM热激后的cDNA为模板,采用反式PCR获得基因5’端序列,应用3’-RACE获得基因3’端序列。再以5’、3’端序列设计引物,PCR扩增获得基因的开放阅读框（ORF）,全长465bp,包含154个氨基酸,命名为RcHSP17.8（登录号:EF053229）。BLAST检索显示它与其它物种小分子热激蛋白基因（sHSP）具有较高同源性,氨基酸序列比对发现其包含胞质Ⅰ类sHSP的特征保守序列,聚类分析显示它位于双子叶植物胞质Ⅰ类sHSP区域,该基因属胞质Ⅰ类sHSP基因。Southern杂交表明该基因在月季基因组中为低拷贝存在。月季RcHSP17.8为温度诱导表达基因,38℃热激5min就能检测转录子,5h后表达量开始下降。热激下,该基因在花中的表达量明显高于其它器官,推测RcHSP17.8可能与花发育有关。此外,NaCl、PEG4000、蔗糖、H₂O₂等胁迫均能诱导RcHSP17.8表达,证明RcHSP17.8参与月季对高盐、高渗、氧化等非生物胁迫的响应。构建原核表达载体pET32a-RcHSP 17.8转化大肠杆菌BL21,SDS-PAGE结果显示,转化菌株在IPTG诱导下0.5h就可检测到含RcHSP17.8的融合蛋白。Western印迹显示在37℃常温下IPTG诱导0.5h转pET32a-RcHSP17.8菌株就表达目的融合蛋白,2h达最大值,6h后表达量开始下降;50℃高温胁迫下,0.5h时转化菌株就开始高表达目的蛋白,5h时还维持很高表达量。上述实验结果表明转化菌株在常温下能正常表达RcHSP17.8的融合蛋白,而高温引起其表达量增高。抗性胁迫实验证明RcHSP17.8的转入提高了大肠杆菌对高温、低温、高盐、高pH、重金属、氧化等非生物胁迫的耐性。构建酵母真核表达载体pPIC3.5K-YEP-RcHSP17.8,经BglⅡ线性化后采用电击法转化酵母SMD1168,通过G418筛选获得多拷贝阳性克隆菌株。共定位显微观察显示YEP只在胞质中表达,结果表明同源重组的酵母能正常表达含有目的基因的融合蛋白,且定位在细胞质,这与RcHSP17.8属胞质Ⅰ类sHSP的推理相吻合。同源重组酵母在50℃高温、4℃低温下的菌落生长状况明显好于空载转化酵母,表明RcHSP17.8基因的转入提高了酵母对高温与低温的耐受性。此外,转化后的酵母提高了对高盐、高pH、重金属、氧化等非生物胁迫的抗性。构建重组植物表达载体PHB-RcHSP17.8,通过农杆菌介导将RcHSP17.8基因转入拟南芥,经潮霉素筛选获得T₃代转基因种子。Western blot结果显示转基因植株高表达RcHSP17.8;对培养基上生长的株系,进行高温、高盐、高渗处理,发现转基因拟南芥平均根长与存活率均优于野生型拟南芥;对盆栽苗进行干旱处理,发现转基因株系具有较强的抗旱性。RT-PCR检测发现,转基因株系在胁迫时高表达RcHSP17.8,同时还发现一些与胁迫相关的基因,如HSP101、渗调蛋白基因（Osmotin）、甜菜碱醛脱氢酶基因（BADH）、脯氨酸合成酶基因（P5CS）、抗坏血酸过氧化物酶基因（APX）相应高或低表达,证明RcHSP17.8在胁迫条件下与HSP101协同行使了分子伴侣功能,并保护了上述蛋白或酶,从而提高了转基因拟南芥多种胁迫抗性。上述研究结果表明,在胁迫条件下,月季RcHSP17.8基因过表达导致其宿主体内RcHSP17.8的积累,从而提高了宿主对高温、低温、高盐、高渗、高pH、重金属、氧化等多种非生物胁迫的耐受性,证明RcHSP17.8参与了多种非生物胁迫的响应。应用反式PCR获得SM与KP的RcHSP17.8基因启动子,序列比对发现它们在顺式调控元件上存在一些差异,具有高温、低温、ABA、真菌病害等诱导顺式作用元件。启动子克隆与序列初步分析旨在探索月季RcHSP17.8响应多种非生物胁迫的分子机理。更多还原

【Abstract】 Rosa chinensis,which belongs to Rosaceae,is an important continuously blossomed ornamental plant.It plays a key role in the garden ornaments for its various genotypes and colors.So more and more attentions were paid on the researches of Rosa chinensis.With the increasing global greenhouse effect,high temperature has become the main restrictive factor for plant growth and development.Therefore,the research on how to enhance the abiotic stress tolerance such as thermotolerance of Rosa chinensis has large significance.Molecular biology researches of Rosa chinensis are based on high quality extracted DNA and RNA,which is affected by high abundant tannin,polysaccharides,and phenols that contained in the plant body of Rosa chinensis.In this study,we extracted high quality DNA from Rosa chinensis through improved CTAB method,and verified that RNAout kit is suitable for extracting RNA from Rosa chinensis.Meanwhile,we had also constructed an effective rapidly subculture system for it.In this work,we selected two Rosa chinensis varieties,Sohloss Mannieim（SM） and Kordes’Perfecta（KP）,which shows different tolerance to high temperature,SM as resistant and Kpas sensitive one.These two varieties of Rosa chinensis were placed at 38℃for 3 hours,and two-dimensional polyacrylamide gel electrophoresis（2D-PAGE） was used to analyze the protein expression pattern of these two varieties.Several spots which only appeared on the SM gel map were found out and the massspectrum and BLAST analysis implied that one of these spots might be a peptide of a small heat shock protein 17.8（sHSP 17.8）.Open reading frame（ORF） of the HSP17.8 gene was attained by homology cloning, inverse-PCR and 3’-RACE.The ORF is a 465bp fragment,and encoding 154 amino acids,and was designated as RcHSP17.8（GeneBank accession number:EF053229）. The deduced amino acid sequence was aligned with other representative sHSPs and demonstrated significant homology to sHSPs of other plants.Phylogenetic analysis revealed RcHSP17.8 to be a cytosolic class I sHSP.RT-PCR revealed that RcHSP17.8 is a high temperature induced expressing gene, and is rapidly induced expressing after 5min heat stress treatment（38℃）,and after 5h, the mRNA level began to decline.RcHSP17.8 is more strongly expressed in flowers than that in other organs of Rosa chinensis under heat stress treatment.In addition, NaCl,PEG4000,sucrose and H₂O₂ also induce the expression of RcHSP17.8.Recombinant RcHSP17.8 was overexpressed in Escherichia coli（BL21） using pET32a,and also in yeast（SMD 1168） by the vector of pPIC3.5K to study its possible function under stress conditions.The recombinant E.coli and yeast cells that contained RcHSP17.8 showed improved viability under different stress conditions, such as high and low temperature,high salt concentration,heavy metal and oxidative stress,as compared with control cultures.Transgenic Arabidopsis thaliana that constitutively expressed RcHSP17.8 exhibited increased tolerance to thermal,salt and osmotic stress.These results suggest that expression of RcHSP17.8 enhances the thermotolerance and other abiotic stress resistance in both prokaryotes and eukaryotes. Besides,expressing of stress-correlative genes in Arabidopsis thaliana such as HSP101,Osmotin,BADH,P5CS and APX are parallel with the expressing of RcHSP 17.8.We also isolated promoter sequences of RcHSP17.8 from two varieties,SM and KP using inverse-PCR.Sequence alignment revealed that special elements existed in the promoter of SM,maybe it is related to regulate the expression of target gene.Some cis-regulate elements,such as HSE,LTR,ABA,MBS existed in both of promoter which are correlative with temperature and ABA stress regulation.更多还原