酵母盐胁迫应答基因的克隆及表达研究

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1、摘要日懿严重的土壤盐渍化使通过遗传工程手段提商植物的耐盐性成为未来环境霹袭鼗浚震霉嚣遥蓊筑先籍凌酌凌题。遥去抟掰究工{管在蓥定移分离一些在穗耪薅盐过程中有潜力的基因方面取得了显著的进展。为了了解盐胁迫应答反应的分子基獭，势寒搦关键靛辩薤基瑗，奉磷究竟黧了盐诱器簿霉ESTs秘簿母瓣簸基蠢HALl，弗对其凝达进行了分析。戮鲁氏酵母蘧(Zgoccharomyces．roffxii)赘椽AS2。i81为实验材料，瑷簸骆遭处理的鹊氏酵母细胞和未经胁迫处理的酵母细胞构建了抑制性消减杂交文摩。运用～耱毫效熬鞋PCR蔽零为基础酾cDNA清减杂交方法构建

2、了铸氏酵母酌盐胁迫应铃差异袭达ESTs文库。随机挑取cDNA克隆进行了测序分栅和序列同源性比较。程掰分辑鼹痔列中我裂部分穿剜与数掭库孛叠知的穿猁有丽源性，其中部分序列与融知序列同源性较高，相应的融知序列与耐盐有关。其余回源性较低的序列以及程数据痒申麓已籍浮弼没脊任旃礴源往酌序鳓可麓是与辩盐福关的新序弼本研究还从酿酒酵母菌菌株AS2．375中克隆了酿溜酵母HALl基照。HALl基灏是簿蹲中重要瓣耐盐纂霹。戳农秆蓠介导韵时圆盘法将HALl基闲转仡烟草植株a筛选刮的转删三J基因转化煅革植椿的耐盐性明显态予对照。通过比较转投旺!纂因龋鼙{壹株与

3、转萁它澍盐基谶烟草植株的耐赭性，发现转赫犯j基溺烟草稹株的耐盐性虽然明显离于对照，但却低于表达渗透调节裁会成酶基缀的转懿困援株。结聚表弱，单独转祀HALl基因对烟草植株耐盐德的提高是有限的。HALl基因可被渗透胁追诱导表达对N矿离子舶搀起重要{乍熙，但对渗透胁迫却无辩瞧，困藤转纯HALl基弱的烟荜植株对盐性的提高是肖限的。渗透胁迫和离予胁迫艇盐胁遗是不W忽视的豫个重要方蘑，默髭珏?基因秘耐渗透胁追基因的复合基因转纯簧路极毒希望使转纂困植物的耐魏性得到大幅度握高。关键谣；辩母；瓣盐；斑答；綦因竞隆；搀铡瞧渣壤杂交；ABSTRACTThep

4、rogressivesalinizationofsoilhasmadethegeneticimprovementofsalttoleranceallurgentpriorityforthefutureofenvironmentandagriculture．Inthelastfewyearssignificantadvanceshavebeenmadeintheidentificationandisolationofseveralgenesthatcouldpotentiallybeinvolvedintheprocessofsaltto

5、lerance．Saltstressadverselyaffectsthegrowthofplantsandmicroorganism，Tounderstandthemolecularbasisofsalt—stressresponseandisolationthekeysalttolerantgene，thesaltinducedESTsandsalttolerantgeneHALlinYeastwereclonedandtheirexpressionwereanalyzed．Suppressivesubstractedhybridi

6、zationeDNAlibrarywasconstructedemployingspecificNaCl一stressedcellsandnon—stressedcellsfromyeaststrain(Zygosaccharomyces．rouxii2．181)。AnefficientPCR—basedeDNAsubstractionmethodwasemployedfortheisolationofthesalt—stressedresponsiveESTs．Cloneswereisolatedrandomlyandsequence

7、d，Theresultsofsequeceanalysisandhomologycomparationshowedthatsomecloneswerehomologoustogenesthathaveearlierbeenimplicatedinstressresponse，othercloneswerenovelwithrespecttotheirfunctionandshowedlowerornohomologytosequencesinthepublicdatabase．SequencesofpartsoftheclonedEST

8、sinthisresearcharenewsequencesinZygosaccharomyces，rouxii。AsalttolerantgeneHALlidentifiedfirstinyeastbyG