拟南芥SPM12基因GFP载体构建及转基因植株筛选
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摘要
[目的]为了进一步研究SPM12基因的功能,利用哥伦比亚(Columbia,Col)遗传背景的野生型拟南芥材料构建SPM12基因GFP载体及其转基因植株。[方法]以野生型拟南芥的RNA反转录成的cDNA为模板,PCR扩增出SPM12基因的CDS全长序列,将其连接到GFP载体的质粒上;然后将构建成功的重组质粒转化到大肠杆菌DH5α受态细胞中,挑取阳性菌落经PCR鉴定并测序后,将其质粒转入农杆菌GV3101感受态细胞中。PCR鉴定出阳性菌落后,通过浸花法将其转入野生型拟南芥植株中。收种子后,使用带有抗性的选择性培养基筛选出阳性植株。[结果]通过SPM12基因CDS片段和SPM12-GFP重组质粒的获得,构建了SPM12-GFP载体和其转基因植株。[结论]成功获得拟南芥SPM12-GFP转基因植株,为进一步研究拟南芥SPM12基因的功能与分子机制奠定了基础。
[Objective] To further study the function of the SPM12 gene, the SPM12 gene GFP vector and its transgenic plants were constructed by wild-type Arabidopsis thaliana with genetic background in Columbia(Columbia, Col). [Method] The full-length CDS sequence of SPM12 gene was amplified by PCR from the cDNA template which was reverse transcription from the RNA of wild-type Arabidopsis thaliana, and ligated it with the plasmid of GFP vector; then transformed the recombinant plasmid which was successfully constructed into the cells of E. coli DH5α, picked the positive colonies and identified it by PCR, and then transformed its plasmid into the competent cells of Agrobacterium GV3101, the positive colonies were identified by PCR, then it was transferred into wild-type Arabidopsis thaliana through inflorescence-infiltrated method. After receiving the seeds, the positive plants were screened by selective medium with resistance. [Result] The CDS fragment of SPM12 gene and SPM12-GFP recombinant plasmid was obtained, the vector of SPM12-GFP and its transgenic plant was got. [Conclusion] The transgenic plants SPM12-GFP in Arabidopsis thaliana were successfully obtained, which laid a foundation for further study of the function and molecular mechanism of SPM12 gene in Arabidopsis thaliana.
[Objective] To further study the function of the SPM12 gene, the SPM12 gene GFP vector and its transgenic plants were constructed by wild-type Arabidopsis thaliana with genetic background in Columbia(Columbia, Col). [Method] The full-length CDS sequence of SPM12 gene was amplified by PCR from the cDNA template which was reverse transcription from the RNA of wild-type Arabidopsis thaliana, and ligated it with the plasmid of GFP vector; then transformed the recombinant plasmid which was successfully constructed into the cells of E. coli DH5α, picked the positive colonies and identified it by PCR, and then transformed its plasmid into the competent cells of Agrobacterium GV3101, the positive colonies were identified by PCR, then it was transferred into wild-type Arabidopsis thaliana through inflorescence-infiltrated method. After receiving the seeds, the positive plants were screened by selective medium with resistance. [Result] The CDS fragment of SPM12 gene and SPM12-GFP recombinant plasmid was obtained, the vector of SPM12-GFP and its transgenic plant was got. [Conclusion] The transgenic plants SPM12-GFP in Arabidopsis thaliana were successfully obtained, which laid a foundation for further study of the function and molecular mechanism of SPM12 gene in Arabidopsis thaliana.
引文
[1] PANDEY S P,SOMSSICH I E.The role of WRKY transcription factors in plant immunity[J].Plant physiology,2009,150(4):1648-1655.
[2] SHI W Y,SHAO H B,LI H,et al.Progress in the remediation of hazardous heavy metal-polluted soils by natural zeolite[J].Journal of hazardous materials,2009,170(1):1-6.
[3] GJORGIEVA D,KADIFKOVA-PANOVSKA T,RUSKOVSKA T,et al.DNA-damage and total antioxidant status in two selected medicinal plants subjected to heavy metal phytotoxicity[C]// Conference on medicinal and aromatic plants of southeast European countries.[s.l.]:[s.n],2012:127-127.
[4] SYTAR O,KUMAR A,LATOWSKI D,et al.Heavy metal-induced oxidative damage,defense reactions,and detoxification mechanisms in plants[J].Acta physiologiae plantarum,2013,35(4):985-999.
[5] SHAW B P,SAHU S K,MISHRA R K.Heavy metal induced oxidative damage in terrestrial plants[M]//PRASAD M N V.Heavy metal stress in plants.Berlin Heidelberg:Springer-Verlag,2004:84-126.
[6] LEE J,BAE H,JEONG J,et al.Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals[J].Plant physiology,2003,133(2):589-596.
[7] ROSS S M.Sources and forms of potentially toxic metal in soil-plant system[M]// ROSS S M.Toxic metals in soil-plant systems.New York:John Wiley & Sons,1994:3-25.
[8] SHAFFER M.Waste lands:The threat of toxic fertilizer[M].Los Angeles,CA:California’s Advocate for the Public Interest,2001.
[9] MENCH M J.Cadmium availability to plants in relation to major long-term changes in agronomy systems[J].Agric Ecosyst Environ,1998,67(2/3):175-187.
[10] RASKIN I,SMITH R D,SALT D E.Phytoremediation of metals:Using plants to remove pollutants from the environment[J].Curr Opin Biotechnol,1997,8(2):221-226.
[11] 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].Plant cell,2009,21:4031-4043.
[12] 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.
[13] GOYER R A.Toxic and essential metal interactions[J].Annu Rev Nutr,1997,17:37-50.
[2] SHI W Y,SHAO H B,LI H,et al.Progress in the remediation of hazardous heavy metal-polluted soils by natural zeolite[J].Journal of hazardous materials,2009,170(1):1-6.
[3] GJORGIEVA D,KADIFKOVA-PANOVSKA T,RUSKOVSKA T,et al.DNA-damage and total antioxidant status in two selected medicinal plants subjected to heavy metal phytotoxicity[C]// Conference on medicinal and aromatic plants of southeast European countries.[s.l.]:[s.n],2012:127-127.
[4] SYTAR O,KUMAR A,LATOWSKI D,et al.Heavy metal-induced oxidative damage,defense reactions,and detoxification mechanisms in plants[J].Acta physiologiae plantarum,2013,35(4):985-999.
[5] SHAW B P,SAHU S K,MISHRA R K.Heavy metal induced oxidative damage in terrestrial plants[M]//PRASAD M N V.Heavy metal stress in plants.Berlin Heidelberg:Springer-Verlag,2004:84-126.
[6] LEE J,BAE H,JEONG J,et al.Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals[J].Plant physiology,2003,133(2):589-596.
[7] ROSS S M.Sources and forms of potentially toxic metal in soil-plant system[M]// ROSS S M.Toxic metals in soil-plant systems.New York:John Wiley & Sons,1994:3-25.
[8] SHAFFER M.Waste lands:The threat of toxic fertilizer[M].Los Angeles,CA:California’s Advocate for the Public Interest,2001.
[9] MENCH M J.Cadmium availability to plants in relation to major long-term changes in agronomy systems[J].Agric Ecosyst Environ,1998,67(2/3):175-187.
[10] RASKIN I,SMITH R D,SALT D E.Phytoremediation of metals:Using plants to remove pollutants from the environment[J].Curr Opin Biotechnol,1997,8(2):221-226.
[11] 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].Plant cell,2009,21:4031-4043.
[12] 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.
[13] GOYER R A.Toxic and essential metal interactions[J].Annu Rev Nutr,1997,17:37-50.