农杆菌介导甘蔗ACC氧化酶基因的遗传转化研究
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摘要
根据已报道的甘蔗ACO基因的序列,在读码框外设计特异引物,通过RT-PCR技术,克隆了甘蔗ACO基因编码区969bp的cDNA片段,分别正向和反向插入植物表达载体pBI121的CaMV35S启动子和NOS终止子之间,构建了ACO基因的正义植物表达载体pBIaco和反义植物表达载体pBIantiaco,该载体的抗性选择标记为NPTⅡ基因。
利用冻融法分别将正义植物表达载体pBIaco和反义植物表达载体pBIantiaco导入根癌农杆菌菌株EHA105中,通过农杆菌介导法,将ACO正、反义基因导入烟草K346。经PCR筛选,获得34株正义和28株反义转基因植株,转化率分别为24%和31%。取部分PCR阳性植株进行Dot-Southern杂交检测,证明外源基因已整合入烟草的基因组中。采用气相色谱法对转基因烟草进行内源乙烯释放量测定,结果显示,正义转基因烟草的乙烯释放比对照平均增加77.22%,面反义转基因烟草的乙烯释放量仅为对照的31.67%,达到极显著差异。说明甘蔗ACO正、反义基因可在异源真核系统中实现功能性表达。转基因烟草移栽后,反义转基因烟草表现出前期生长延缓、后期开花延迟特性,正义转基因烟草表现出前期生长快、后期开花早特性。
通过农杆菌介导法将ACO反义基因遗传转化甘蔗ROC22,经过G418抗性筛选,获得19株抗性植株。对选择标记NPTⅡ基因进行PCR检测,有2株呈现阳性,该阳性转基因植株生长状况较野生型和PCR阴性的植株差,生长迟缓,矮化。
Primers were designed according to the reported sequence of ACO gene in sugarcane.A 969bp fragment of ACC oxidase was amplified from cDNA of sugarcane through RT-PCR and then cloned.The cloned ACC oxidase gene was then inserted into a binary vector pBI121 with the forward or reverse orientation between the CaMV35S promoter and NOS terminator sequence respectively,to construct the sense plant expression vector pBIaco and the antisense plant expression vector pBIantiaco,both contained a Km resistance gene(NPTⅡ).
The recombinant vectors of pBIaco and pBIantiaco were introduced into Agrobacterium tumefaciens strain EHA105 with freeze-thaw method, respectively.Then the leaf discs of tobacco plants were transformed with ACO gene and ACO antisense gene.34 sense transgenic plants and 28 antisense transgenic plants had been confirmed by PCR analysis,with the transformation efficiency of 24%and 31%,respectively.Parts of regenerated kanamycin-resistant plants were selected and proven to be positive by Dot-Southern bloting,indicating that the ACO gene and the antisense gene had been integrated into tobacco genome.The ethylene production from leaves of transgenic and nontransgenic tobacco were determined by gas chromatography(GC).The ethylene productions from ACO sense transgenic plants was increased by 77.22%,and that from ACO antisense transgenic plants was reduced by 68.33%,respectively,in compared with that from nontransgenic plantlet.The results confirmed the expression of sugarcane ACO gene in heterologous eukaryotic system.The tobacco transformed with sense ACO grew faster and flowered earlier than the antisense transformers.
Subsequently,sugarcane ROC22 was transformed with ACO antisense gene mediated by Agrobacterium tumefaciens.The transformed shoots were screened under the selective pessure of G418 and 19 resistants were obtained, and two of which were further proven to be positive through detecting NPTⅡby PCR.The positive plants grew slowlier and were dwarf than the wild type and the untransformed sugarecane as well.
利用冻融法分别将正义植物表达载体pBIaco和反义植物表达载体pBIantiaco导入根癌农杆菌菌株EHA105中,通过农杆菌介导法,将ACO正、反义基因导入烟草K346。经PCR筛选,获得34株正义和28株反义转基因植株,转化率分别为24%和31%。取部分PCR阳性植株进行Dot-Southern杂交检测,证明外源基因已整合入烟草的基因组中。采用气相色谱法对转基因烟草进行内源乙烯释放量测定,结果显示,正义转基因烟草的乙烯释放比对照平均增加77.22%,面反义转基因烟草的乙烯释放量仅为对照的31.67%,达到极显著差异。说明甘蔗ACO正、反义基因可在异源真核系统中实现功能性表达。转基因烟草移栽后,反义转基因烟草表现出前期生长延缓、后期开花延迟特性,正义转基因烟草表现出前期生长快、后期开花早特性。
通过农杆菌介导法将ACO反义基因遗传转化甘蔗ROC22,经过G418抗性筛选,获得19株抗性植株。对选择标记NPTⅡ基因进行PCR检测,有2株呈现阳性,该阳性转基因植株生长状况较野生型和PCR阴性的植株差,生长迟缓,矮化。
Primers were designed according to the reported sequence of ACO gene in sugarcane.A 969bp fragment of ACC oxidase was amplified from cDNA of sugarcane through RT-PCR and then cloned.The cloned ACC oxidase gene was then inserted into a binary vector pBI121 with the forward or reverse orientation between the CaMV35S promoter and NOS terminator sequence respectively,to construct the sense plant expression vector pBIaco and the antisense plant expression vector pBIantiaco,both contained a Km resistance gene(NPTⅡ).
The recombinant vectors of pBIaco and pBIantiaco were introduced into Agrobacterium tumefaciens strain EHA105 with freeze-thaw method, respectively.Then the leaf discs of tobacco plants were transformed with ACO gene and ACO antisense gene.34 sense transgenic plants and 28 antisense transgenic plants had been confirmed by PCR analysis,with the transformation efficiency of 24%and 31%,respectively.Parts of regenerated kanamycin-resistant plants were selected and proven to be positive by Dot-Southern bloting,indicating that the ACO gene and the antisense gene had been integrated into tobacco genome.The ethylene production from leaves of transgenic and nontransgenic tobacco were determined by gas chromatography(GC).The ethylene productions from ACO sense transgenic plants was increased by 77.22%,and that from ACO antisense transgenic plants was reduced by 68.33%,respectively,in compared with that from nontransgenic plantlet.The results confirmed the expression of sugarcane ACO gene in heterologous eukaryotic system.The tobacco transformed with sense ACO grew faster and flowered earlier than the antisense transformers.
Subsequently,sugarcane ROC22 was transformed with ACO antisense gene mediated by Agrobacterium tumefaciens.The transformed shoots were screened under the selective pessure of G418 and 19 resistants were obtained, and two of which were further proven to be positive through detecting NPTⅡby PCR.The positive plants grew slowlier and were dwarf than the wild type and the untransformed sugarecane as well.
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