拟南芥MDH2基因GFP载体构建及转基因植株的筛选鉴定
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摘要
[目的]以野生型拟南芥为材料构建MDH2基因GFP载体及GFP转基因植株,研究MDH2基因在植物响应镉胁迫机制中的功能。[方法]通过提取野生型拟南芥的RNA,反转录成为cDNA,并以cDNA为模板,利用PCR扩增MDH2基因,将扩增所获得MDH2基因和pXB94-GFP质粒双酶切后连接,并转化进大肠杆菌感受态细胞中,鉴定正确后再转入农杆菌感受态细胞中,通过菌落PCR鉴定出阳性单菌落。再通过浸花法转入野生型拟南芥中,最后利用抗性筛选和PCR鉴定获得MDH2转基因阳性植株。[结果]成功克隆MDH2基因,并构建了MDH2-GFP重组质粒,抗性筛选获得了MDH2-GFP转基因植株。[结论]成功获得MDH2-GFP转基因植株,为进一步研究该基因在植物响应镉胁迫机制中的功能奠定了基础。
[Objective] In order to further study the function of MDH2 gene in response to cadmium stress in plant,the vector of Arabidopsis MDH2-GFP was built up and transgenic lines were constructed.[Method] The RNA of wild-type Arabidopsis was extracted and reverse transcribed into cDNA,the full-length MDH2 coding sequence was amplified by PCR. The amplified MDH2 gene and pXB94-GFP plasmid were digested and connected by double enzymes.The connected product was transformed into E.coli competent cells,then the true recombinant plasmid was transformed into Agrobacterium tumefaciens competent cells and the positive single colony was identified by colony PCR,then it was transferred into wild-type Arabidopsis by the floral-dip method.Finally,the positive transgenic plants were obtained by resistance screening and PCR identification.[Result]MDH2 gene was successfully cloned and the positive transgenic lines were obtained by selective medium with resistance.[Conclusion]The successful acquisition of MDH2-GFP transgenic lines laid the foundation for further research on the function of MDH2 gene.
[Objective] In order to further study the function of MDH2 gene in response to cadmium stress in plant,the vector of Arabidopsis MDH2-GFP was built up and transgenic lines were constructed.[Method] The RNA of wild-type Arabidopsis was extracted and reverse transcribed into cDNA,the full-length MDH2 coding sequence was amplified by PCR. The amplified MDH2 gene and pXB94-GFP plasmid were digested and connected by double enzymes.The connected product was transformed into E.coli competent cells,then the true recombinant plasmid was transformed into Agrobacterium tumefaciens competent cells and the positive single colony was identified by colony PCR,then it was transferred into wild-type Arabidopsis by the floral-dip method.Finally,the positive transgenic plants were obtained by resistance screening and PCR identification.[Result]MDH2 gene was successfully cloned and the positive transgenic lines were obtained by selective medium with resistance.[Conclusion]The successful acquisition of MDH2-GFP transgenic lines laid the foundation for further research on the function of MDH2 gene.
引文
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[2] SHIM D,HWANG J U,LEE J,et al.Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice[J].The plant cell,2009,21(12):4031-4043.
[3] GALLEGO S M,PENA L B,BARCIA R A,et al.Unravelling cadmium toxicity and tolerance in plants:Insight into regulatory mechanisms[J].Environ Exp Bot,2012,83:33-46.
[4] STRAIF K,BENBRAHIM-TALLAA L,BAAN R,et al.A review of human carcinogens-Part C:Metals,arsenic,dusts,and fibres[J].Lancet Oncol,2009,10(5):453-454.
[5] KIM D Y,BOVET L,KUSHNIR S,et al.AtATM3 is involved in heavy metal resistance in Arabidopsis[J].Plant Physiol,2006,140(3):922-932.
[6] HALL J L.Cellular mechanisms for heavy metal detoxification and tolerance[J].J Exp Bot,2002,53:1-11.
[7] CLEMENS S.Molecular mechanisms of plant metal tolerance and homeostasis[J].Planta,2001,212(4):475-486.
[8] BELHAJ K,CHAPARRO-GARCIA A,KAMOUN S,et al.Plant genome editing made easy:Targeted mutagenesis in model and crop plants using the CRISPR/Cas system[J].Plant methods,2013,9(1):1-10.
[9] NAWROT T,PLUSQUIN M,HOGERVORET J,et al.Environmental exposure to cadmium and risk of cancer:A prospective population-based study [J].Lancet Oncol,2006,7(2):119-126.
[10] LU L L,TIAN S K,YANG X E,et a1.Cadmium uptake and xylem loading are active processes in the hyperaccumulator Sedum alfredii[J].Joumal of plant physiology,2009,166(6):579-587.