拟南芥bZIP23基因过量表达载体的构建及过表达植株的筛选_顾菊.pdf

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1、DOI:10.13989/j.cnki.0517-6611.2014.14.007安徽农业科学，JournalofAnhuiAgri．Sci．2014，42(14):4199－4201责任编辑石金友责任校对李岩拟南芥bZIP23基因过量表达载体的构建及过表达植株的筛选*顾菊，韩娇，董万春，阳立波，曹树青(合肥工业大学生物与食品工程学院，安徽合肥230009)摘要［目的］以拟南芥为材料克隆bZIP23基因，构建bZIP23基因的过量表达载体和筛选过表达植株，为验证其功能奠定基础。［方法］提取拟南芥总ＲNA和ＲT-PCＲ克隆bZIP23基因，

2、用限制性内切酶切割和T4DNA连接酶连接，使bZIP23基因连接到35S强启动子的pAＲT27载体上;将连接产物转化到Trans1-T1感受态细胞中，筛选阳性单克隆进行菌落PCＲ鉴定并测序验证，获得重组质粒。将该重组质粒电激转化至根瘤农杆菌GV3101菌株，浸花法转化拟南芥野生型植株。［结果］通过单菌落PCＲ鉴定和DNA测序结果显示，bZIP23基因与35S过量表达载体已连接，获得了重组载体;抗性筛选与遗传鉴定获得相应的转基因过量表达阳性植株。［结论］构建的过量表达载体及筛选得到的过量表达植株为验证bZIP23基因功能奠定了基础。关键词拟

3、南芥(Arabidopsisathaliana);bZIP23;互补和过量表达载体;转基因植株中图分类号Q754;S188文献标识码A文章编号0517－6611(2014)14－04199－03ConstructionofArabidopsisGenebZIP23OverexpressionVectorandScreeningofExpressionPlantGUJu，CAOShu-qingetal(SchoolofBiotechnologyandFoodEngineering，HefeiUniversityofTechnology，He

4、fei，Anhui230009)Abstract［Objective］ArabidopsiswereusedasmaterialtoclonebZIP23gene，weconstructgenebZIP23overexpressionvectorandscreenex-pressionplant．［Method］TotalＲNAwasextractedfromArabidopsisseedlings，andcDNAfragmentsofbZIP23genewereamplifiedbyＲT-PCＲ．Usingtherestrictione

5、nzymesandT4DNAligase，cDNAfragmentsweresubsequentlyclonedintoPAＲT27vectors，andthenweretransformedintoTrans-T1phageresistantchemicallycompetentcells．AnalysisofbacterialcolonyPCＲandcDNAsequencingwereperformedtoconfirmthatcD-NAoftheArabidopsisthalianabZIP23genewassuccessfully

6、cloned．TherecombinantplasmidswereobtainedandtransformedintoAgrobacteriumGV3101cells．Wild-typeArabidopsisthalianawastransformedbyusingfloral-dipmethod．［Ｒesult］AnalysisofbacterialcolonyPCＲandDNAsequencingwereperformedtoconfirmrecombinantplasmids，complementaryandoverexpressi

7、onpositiveplantswereobtainedthroughgeneticscreeningandidentificationofgeneticallymodifiedmethods．［Conclusion］ConstructionofArabidopsisgenebZIP23overexpressionvectorandscreeningofexpressionplantlaidthefoundationforthefunctionofgenebZIP23．KeywordsArabidopsisthaliana;bZIP23;

8、Complementaryandoverexpressionvector;Geneticallymodifiedplant转录因子是一类识别DNA特异序列且结合在目的基因繁衍保存。启动子的特定