苏云金芽胞杆菌抗软腐病aiiA基因转花魔芋研究

Regeneration of Transgenic Amorphophallus konjac Expressing Synthesized acyl-homoserine lactonase (aiiA) gene from Bacillus thuringiensis

采用农杆菌介导的遗传转化方法,将来自苏云金芽胞杆菌经密码子优化的人工合成酰基高丝氨酸环内酯酶aiiA基因导入花魔芋,以提高魔芋抗软腐病的能力。以花魔芋的茎为外植体,通过与含有植物双元表达载体pU1301的农杆菌EHA105共培养,将aiiA基因导入魔芋基因组。经过潮霉素两次筛选,得到9块独立的抗性愈伤组织,经分化,生根,移入温室盆栽成活的抗性再生苗34株。对不同抗性愈伤来源的T0代再生苗进行PCR检测得到的阳性植株比例为62.7%,斑点杂交进一步确认外源aiiA基因已成功地整合到魔芋基因组中。Western杂交显示,aiiA基因在魔芋植株中能正常表达,其最高表达量占魔芋叶片总可溶蛋白的0.02%～0.06%。酰基高丝氨酸环内酯酶活性检测表明转基因魔芋叶片的蛋白可以降解酰基高丝氨酸环内酯信号分子。

An expression vector pU1301-aiiA containing a synthesized gene encoding acyl-homoserine lactonase （aiiA） gene from Bacillus thuringiensis driven by maize ubiquitin promoter was constructed and then transferred into A morphophallus konjac mediated by Agrobacterium tumefaciens. Only 9 resistant calli were produced, finally 34 resistant plants were obtained. The independented plantlets regenerated from different calli were assayed by PCR. The positive rate of PCR was 62.7 % in all putative transgenic plants. Dot hybridization showed that the target gene was integrated into the genome of Amorphophallus konjac. Western blotting demonstrated the expressed protein of interest being reactive with the antibody against AliA. The levels of AliA expression were up to 0.02%-0.06% of total soluble Amorphophallus konjac leaf protein. Expressed protein of traasgenic A morphophallus konjac plants was analyzed by detection of AiiA activity, which had the same bioactivity as the natural AiiA protein.