《酵母鹽脅迫應(yīng)答基因的克隆及表達(dá)研究》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。

1、摘要日懿嚴(yán)重的土壤鹽漬化使通過遺傳工程手段提商植物的耐鹽性成為未來環(huán)境霹襲鼗浚震霉囂遙蓊筑先籍凌酌凌題。遙去摶掰究工{管在鎣定移分離一些在穗耪薅鹽過程中有潛力的基因方面取得了顯著的進(jìn)展。為了了解鹽脅迫應(yīng)答反應(yīng)的分子基獺，勢(shì)寒搦關(guān)鍵靛辯薤基瑗，奉磷究竟黧了鹽誘器簿霉ESTs秘簿母瓣簸基蠢HALl，弗對(duì)其凝達(dá)進(jìn)行了分析。戮魯氏酵母蘧(Zgoccharomyces．roffxii)贅椽AS2。i81為實(shí)驗(yàn)材料，璦簸駱遭處理的鵲氏酵母細(xì)胞和未經(jīng)脅迫處理的酵母細(xì)胞構(gòu)建了抑制性消減雜交文摩。運(yùn)用～耱毫效熬鞋PCR蔽零為基礎(chǔ)釃cDNA清減雜交方法構(gòu)建

2、了鑄氏酵母酌鹽脅迫應(yīng)鈴差異襲達(dá)ESTs文庫。隨機(jī)挑取cDNA克隆進(jìn)行了測(cè)序分柵和序列同源性比較。程掰分輯鼴痔列中我裂部分穿剜與數(shù)掭庫孛疊知的穿猁有麗源性，其中部分序列與融知序列同源性較高，相應(yīng)的融知序列與耐鹽有關(guān)。其余回源性較低的序列以及程數(shù)據(jù)癢申麓已籍浮弼沒脊任旃礴源往酌序鰳可麓是與辯鹽福關(guān)的新序弼本研究還從釀酒酵母菌菌株AS2．375中克隆了釀溜酵母HALl基照。HALl基灝是簿蹲中重要瓣耐鹽纂霹。戳農(nóng)稈蘺介導(dǎo)韻時(shí)圓盤法將HALl基閑轉(zhuǎn)仡煙草植株a篩選刮的轉(zhuǎn)刪三J基因轉(zhuǎn)化煅革植椿的耐鹽性明顯態(tài)予對(duì)照。通過比較轉(zhuǎn)投旺!纂因齲鼙{壹株與

3、轉(zhuǎn)萁它澍鹽基讖煙草植株的耐赭性，發(fā)現(xiàn)轉(zhuǎn)赫犯j基溺煙草稹株的耐鹽性雖然明顯離于對(duì)照，但卻低于表達(dá)滲透調(diào)節(jié)裁會(huì)成酶基綴的轉(zhuǎn)懿困援株。結(jié)聚表弱，單獨(dú)轉(zhuǎn)祀HALl基因?qū)煵葜仓昴望}德的提高是有限的。HALl基因可被滲透脅追誘導(dǎo)表達(dá)對(duì)N礦離子舶攙起重要{乍熙，但對(duì)滲透脅迫卻無辯瞧，困藤轉(zhuǎn)純HALl基弱的煙蓽植株對(duì)鹽性的提高是肖限的。滲透脅迫和離予脅迫艇鹽脅遺是不W忽視的豫個(gè)重要方蘑，默髭玨?基因秘耐滲透脅追基因的復(fù)合基因轉(zhuǎn)純簧路極毒希望使轉(zhuǎn)纂困植物的耐魏性得到大幅度握高。關(guān)鍵謠；辯母；瓣鹽；斑答；綦因競(jìng)??；攙鍘瞧渣壤雜交；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