拟南芥MYB4基因GUS载体构建及其转基因植株的筛选
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摘要
[目的]进一步验证MYB4基因在响应干旱胁迫中的应答功能,构建ProMYB4:GUS载体,通过筛选鉴定获得相应的转基因植株。[方法]以野生型拟南芥植株的全基因组为模板,利用特异性引物扩增MYB4基因启动子,将目的基因连接到pART27载体上。然后将构建成功的重组载体转化至农杆菌GV3101,浸花法转化野生型植株。最后通过抗性筛选和PCR鉴定获得阳性转基因植株。[结果]MYB4启动子成功克隆,测序结果经过比对完全正确。抗性筛选获得了阳性转基因植株。[结论]成功获得ProMYB4:GUS阳性转基因植株,为进一步研究MYB4基因功能奠定了基础。
[Objective]To further study the function of MYB4 gene in response to drought stress in plant,the vector of ProMYB4:GUS was constructed,and the corresponding transgenic plants were obtained through screening and identification.[Method]The promoter of MYB4 gene was amplified through PCR from the whole-genome of Arabidopsis thaliana(wild-type) using specific primers and cloned into pART27 vector.And then the recombinant vector was transformed into Agrobacterium strain GV3101 for transformation into wild-type A.thaliana by the floral-dip method.Finally,the positive transgenic plants were obtained by resistance screening and PCR identification.[Result]We succeeded in cloning the promoter of MYB4 gene,the sequencing results were completely correct.Positive transgenic plants were obtained by resistance screening.[Conclusion]The successful acquisition of ProMYB4:GUS transgenic lines laid the foundation for further research on the function of MYB4 gene.
[Objective]To further study the function of MYB4 gene in response to drought stress in plant,the vector of ProMYB4:GUS was constructed,and the corresponding transgenic plants were obtained through screening and identification.[Method]The promoter of MYB4 gene was amplified through PCR from the whole-genome of Arabidopsis thaliana(wild-type) using specific primers and cloned into pART27 vector.And then the recombinant vector was transformed into Agrobacterium strain GV3101 for transformation into wild-type A.thaliana by the floral-dip method.Finally,the positive transgenic plants were obtained by resistance screening and PCR identification.[Result]We succeeded in cloning the promoter of MYB4 gene,the sequencing results were completely correct.Positive transgenic plants were obtained by resistance screening.[Conclusion]The successful acquisition of ProMYB4:GUS transgenic lines laid the foundation for further research on the function of MYB4 gene.
引文
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[6] HEPWORTH C,DOHENY-ADAMS T,HUNT L,et al.Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake[J].New Phytol,2015,208(2):336-341.
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[8] BRAUN E L,GROTEWOLD E.Newly discovered plant c-myb like genes rewrite the evolution of the plant myb gene family[J].Plant Physiol,1999,121(1):21-24.
[9] BERGMANN D C,SACK F D.Stomatal development[J].Annu Rev Plant Biol,2007,58:163-181.
[10] CHAVES M M,MAROCO J P,PEREIRA J S.Understanding plant responses to drought-from genes to the whole plant[J].Funct Plant Biol,2003,30:239-264.
[11] 彭立新,李德全,束怀瑞.园艺植物水分胁迫生理及耐旱机制研究进展[J].西北植物学报,2002,22(5):1275-1281.
[12] LAWLOR D W.Genetic engineering to improve plant performance under drought:Physiological evaluation of achievements,limitations,and possibilities[J].J Exp Bot,2013,64(1):83-108.
[13] WANG H C,NGWENYAMA N,LIU Y D,et al.Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis[J].Plant cell,2007,19(1):63-73.
[2] RENA A B,SPLITTSTOESSER W E.Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons[J].Phytochemistry,1975,14:657-661.
[3] 李玲,余光辉,曾富华.水分胁迫下植物脯氨酸累积的分子机理[J].华南师范大学学报(自然科学版),2003(1):126-134.
[4] DOCIMO T,MATTANA M,FASANO R,et al.Ectopic expression of the Osmyb4 rice gene enhances synthesis of hydroxycinnamic acid derivatives in tobacco and clary sage[J].Biol Plant,2013,57(1):179-183.
[5] BLUM A.Genomics for drought resistance-getting down to earth [J].Functional plant biology,2014,41(11):1191-1198.
[6] HEPWORTH C,DOHENY-ADAMS T,HUNT L,et al.Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake[J].New Phytol,2015,208(2):336-341.
[7] NAKABAYASHI R,YONEKURA-SAKAKIBARA K,URANO K,et al.Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids[J].Plant J,2014,77(3):367-379.
[8] BRAUN E L,GROTEWOLD E.Newly discovered plant c-myb like genes rewrite the evolution of the plant myb gene family[J].Plant Physiol,1999,121(1):21-24.
[9] BERGMANN D C,SACK F D.Stomatal development[J].Annu Rev Plant Biol,2007,58:163-181.
[10] CHAVES M M,MAROCO J P,PEREIRA J S.Understanding plant responses to drought-from genes to the whole plant[J].Funct Plant Biol,2003,30:239-264.
[11] 彭立新,李德全,束怀瑞.园艺植物水分胁迫生理及耐旱机制研究进展[J].西北植物学报,2002,22(5):1275-1281.
[12] LAWLOR D W.Genetic engineering to improve plant performance under drought:Physiological evaluation of achievements,limitations,and possibilities[J].J Exp Bot,2013,64(1):83-108.
[13] WANG H C,NGWENYAMA N,LIU Y D,et al.Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis[J].Plant cell,2007,19(1):63-73.