反馈抑制不敏感邻氨基苯甲酸合成酶基因作为筛选标记基因用于大豆遗传转化研究(英文)  

Feedback-insensitive Anthranilate Synthase Gene as a Novel Selectable Marker for Soybean Transformation

作　　者：陈士云[1] 

机构地区：[1]中国科学院武汉病毒研究所,武汉430071

出　　处：《生物工程学报》2004年第5期646-651,共6页Chinese Journal of Biotechnology

摘　　要：目前广泛采用的抗菌素或抗除草剂基因作为植物转化筛选标记基因可能带来转基因逃逸 ,因此寻找能够用于植物转化的来源于植物本身的筛选基因是解决这一问题的方法之一。通过从烟草中克隆的邻氨基苯甲酸合成酶基因 (ASA2 )作为筛选标记基因 ,并采用氨基酸的类似物 5 甲基色氨酸为筛选剂 ,进行了农杆菌介导的大豆成熟胚尖转化研究。Southern杂交结果表明ASA2基因成功整合到大豆基因组 ,Northern杂交也显示该基因在转化大豆叶片中表达。HPLC检测转化大豆叶片游离色氨酸的含量比野生型要高 5 9%～ 12 3%。PCR检测转化子 1代结果显示转化基因通过孟德尔规律稳定遗传。这些结果表明反馈抑制不敏感ASA2基因可以作为筛选标记基因用于大豆遗传转化。同时也证实来源于一种植物 (烟草 )编码的邻氨基苯甲酸α 亚基能够与另一种植物 (大豆 )编码该酶的 β 亚基结合形成具有完整活性的邻氨基苯甲酸合成酶。Because of the concern about escape of antibiotic or herbicide resistant transgenes from transgenic crops, selectable marker genes from plant origin would be an alternative choice for plant transformation. In this study, a feedback insensitive anthranilate synthase gene ( ASA2 ) cloned from a tobacco cell line was tested for Agrobacterium mediated transformation of axis tissue of soybean mature embryo, with a tryptophan analogue 5 methyltryptophan (5 MT) as the selective agent. Southern blot analysis of the T 0 transgenic lines confirmed the integration of the ASA2 gene into the soybean genome. Northern blot analysis showed the ASA2 gene was also expressed in the leave tissue, and the free tryptophan content in the leaf tissue of transgenic soybean was about 59% to 123% more than that in the wild type. PCR analysis of the T 1 progeny showed that the transgene was inherited in a Mendelian fashion. All these results indicate that this feedback insensitive ASA2 gene can be used as a selectable marker gene for plant transformation. This work also demonstrated that the ASA2 gene coding for the α subunits from one plant (tobacco) can interact with the β subunits of a heterologous plant (soybean) to form an active anthranilate synthase enzyme. The use of this feedback insensitive gene as a novel selectable marker for plant transformation is also discussed.

关 键 词：邻氨基苯甲酸合成酶 反馈抑制不敏感 筛选标记 基因 5-甲基色氨酸 大豆 遗传转化 

分 类 号：S565.1[农业科学—作物学]