本研究建立了小黑杨(Populus simonii×P .nigra)组培再生体系,确定小黑杨芽分化最适宜培养基、生根培养基,然后进行卡那霉素敏感性试验。以小黑杨无菌苗叶片为转化受体材料,通过根癌农杆菌(Agrobacterium tumefacien)介导法将外源基因Bet-A(编码胆碱脱氢酶,催化胆碱生成甘氨酸甜菜碱)导入小黑杨(P.simonii×P.nigra)。对获得的11株转化苗进行了PCR扩增检测和斑点杂交检测,其中7株获得了1.7Kb特异性扩增条带和杂交斑点,表现为阳性。从斑点杂交检测的转化苗中选取3株杂交斑点明亮的转化子进行Southern印迹杂交分析,结果证实外源基因已整合到小黑杨基因组中。
In this study,exogenous gene Bet-A (encoding choline dehydrogenase,synthesizing glycine betaine) was introduced into Populus simonii×P.nigra by Agro-bacterium tumefacien,so as to enhance salt resistance in transformed plant.Firstly,the optimal media of P.simonii×P.nigra for bud differentiation and rooting were established.The sensitivity of the explants to Kanamycin was tested in order to decide a reasonable selective pressure for transformation.Secondly,Bet-A gene was introduced into leaves of asepsis plants of poplar by agrobacterium,and then transformed poplar plants were obtained.PCR and southern blotting analyses showed that Bet-A gene has been integrated into the genome of P.simonii×P.nigra.
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