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1、菠菜甜菜碱醛脱氢酶基因在棉花中的过量表达和抗冻耐逆性分析罗晓丽1,肖娟丽1,王志安1,张安红1,田颖川2,吴家和1,21山西省农业科学院棉花研究所,运城0440002中国科学院微生物研究所植物基因组国家重点实验室,北京100101摘要:分离出菠菜甜菜碱醛脱氢酶基因(SoBADH)构建成由CaMV35S驱动的双元植物表达载体pBSB,农杆菌菌株LBA4404携带该载体转化棉花,获得转基因棉花植株。65株转基因植株经过PCR筛选、Southernblotting分析证明有45株为成功的转化株,外源基因已经被整合到棉花的染色体组中,并以单拷贝插入居多。对部分株系的SoBADH基
2、因的表达进行分析表明均有较高的mRNA和蛋白的表达。经测定这些株系中的甜菜碱脱氢酶活性显著提高,达到0.66~1.70nmol/min/mg水平。同时这些转基因株系在盐胁迫下比对照长势强,株高和地上部分的鲜重显著高于非转基因对照;在低温胁迫下,这些转基因株系表现出显著的抗冻性能。结果表明菠菜甜菜碱醛脱氢酶能够在异源植物棉花中过量表达,并具有较高的酶活性,转基因棉花可作为抗逆育种的种质材料。关键词:棉花,菠菜甜菜碱醛脱氢酶,耐盐性,转基因OverexpressionofSpinaciaoleraceaBetaineAldehydeDehydrogenase(SoBADH)G
3、eneConferstheSaltandColdTolerantinGossypiumhirsutumL.XiaoliLuo1,JuanliXiao1,ZhianWang1,AnhongZhang1,YingchuanTian2,andJiaheWu1,21InstituteofCottonResearch,ShanxiAcademyofAgriculturalScience,Yuncheng044000,China2StateKeyLaboratoryofPlantGenomics,InstituteofMicrobiology,ChineseAcademyofScie
4、nces,Beijing100101,ChinaAbstract:TheopenreadingframeofSpinaciaoleraceaBetaineAldehydeDehydrogenase(SoBADH)wasretrievedfromSpinaciaoleraceaandinsertedintotheAgrobacteriumtumefaciensbinaryvectorpBin438,whichwasdrivenbyCaMV35Spromoter,andproducedthenewbinaryvectorpBSB.A.tumefaciensLBA4404car
5、ryingthisplasmidwasusedingenetictransformationofplants.Forty-fiveprimarytransgenicplantsweredetectedbyPCRandverifiedbytheSouthernblottingfrom65regeneratedplants,ofwhich27transgenicplantshadonlyonecopyofT-DNA.TheNorthernblottingandWesternblottinganalysisindicatedthattheSoBADHgenehadbeentra
6、nscribedmRNAandexpressionproteininthetransgeniccottonlines.ThetestingofSoBADHactivityoftransgenicplantleavesshowedthattheenzymeactivitywasmuchhigherthanthatofthenon-transgeniccotton.Thegrowthoftransgenicplantswaswellunderthesalinityandfreezingstress,whereasthenon-transgenicplantgrewpoorly
7、andevendied.Challengingwithsalinity,theheightandfreshweightoftransgenicplantswashighercomparedwiththoseofnon-transgenicplants.Underthefreezingstress,therelativeconductivityofleafelectrolyteleakageofthetransgeniccottonlineswaslowerthanthatofnon-transgenicplants.These
