农杆菌介导转录因子DREB1C基因转化速生型刺槐的研究

Transformation of transcription factor DREB1C gene into the fast-growing black locust mediated with Agrobacterium tumefaciens.

引自匈牙利的刺槐是可用于观赏、水土保持、饲料和蜂源的速生型树种,向这个树种转入耐逆基因,可满足干旱、盐碱地植被恢复的需求.该文对速生型刺槐进行了3个方面的研究：在含有不同组合6-BA和NAA的MS培养基上的芽再生情况、根癌农杆菌介导GUS（载体为pCAMBIA 1301）基因转化的优化以及耐逆转录因子基因AtDREB1C（相同载体）的转化.结果表明：①以无菌苗复叶叶轴为外植体,在附加6-BA 1.0 mg/L和NAA 0.5 mg/L的MS培养基上芽再生率最高,为77.5%;②同样的外植体在MS培养基上预培养0～2 d后,用附加乙酰丁香酮的根癌农杆菌菌液侵染15～20 min,再接种于附加20 mg/L乙酰丁香酮和Hyg 7 mg/L的上述MS培养基上,共培养2 d,可获得最佳转化效果;③用上述优化的方法将AtDREB1C导入刺槐,获得转基因植株,并得到PCR、PCR-Southernblotting和Southern blotting的验证.

Robinia pseudocacia,introduced from Hungary,is a fast-growing variety used in ornamental,soil and water conservation,fodder and nectar sources.Transformation of this woody plant using abiotic-stress-tolerant gene could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration.In this paper,three aspects of R.pseudocacia were studied： shoot regeneration on MS medium with different combinations of 6-BA and NAA,optimization of β-glucuronidase（GUS） gene（carried by vector pCAMBIA 1301,and mediated by Agrobacterium tumefaciens） transformation,and gene transformation using the transcription factor AtDREB1C gene （abiotic-stress-tolerant gene carried by the same vector）.Results showed that： 1） The highest shoot-regeneration rate 77.5% was obtained from the rachis of compound leaves cultured on MS base medium combimed with 6-BA 1.0 mg/L and NAA 0.5 mg/L;2） The best transformation efficiency was proved by the rachis of compound leaves proculturing for 0—2 days on the MS medium,immerging into the solution containing Agrobacterium and acetosyingone（AS） for 15-20 min,subsequencely co-culturing for two days on the same medium with AS 20 mg/L and hygromycin（Hyg） 7 mg/L;3） Following the method optimized,the AtDREB1C gene was introduced into R.pseudocacia,and transgenic plants were obtained and verified by PCR,PCR-Southern blotting and Southern blotting analysis.