农杆菌介导小球藻快速转化方法的建立与条件优化

Establishment and conditions optimization of Agrobacterium tumefaciens-mediated transformation in Chlorella pyrenoidosa

基于根瘤农杆菌(Agrobacterium tumefaciens)介导的方法实现异养蛋白核小球藻快速遗传转化,并优化转化条件。结果表明,小球藻异养生长条件的碳氮组合配比为葡萄糖20 g/L和酵母粉2 g/L时,生长速率为自养的3~5倍;根瘤农杆菌GV3101和LBA4404均可成功实现对异养小球藻的遗传转化,且转化效率无显著性差异(P>0.05)。最优转化条件为根瘤农杆菌GV3101初始OD600 nm值=0.8,小球藻(Chlorella pyrenoidosa)浓度为107个/mL,各取100μL混合涂布在pH=5.4、乙酰丁香酮浓度为200μmol/L的CM平板上,24℃避光培养40 h后转到筛选平板,仅2 d转化子便清晰可见,数目达88个/106微藻。转化过程整体耗时仅5 d,普遍比自养微藻转化时间缩短3倍以上,为小球藻代谢工程改造提供技术手段,同时为其他可异养培养微藻的短时高效遗传转化提供科学依据。

Based on the method of Agrobacterium tumefaciens-mediated transformation,the method of rapid genetic transformation of heterotrophic pyrenoid Chlorella pyrenoidosa was established and the transformation conditions were optimized.As a result,glucose 20 g/L and yeast extract 2 g/L were selected as the optimal carbon and nitrogen sources for heterotrophic culture of C.pyrenoidosa,respectively,and the growth rate was 3-5 times higher than that of autotrophic culture.A.tumefaciens GV3101 and LBA4404 could successfully achieve the genetic transformation of heterotrophic C.pyrenoidosa without significant difference in transformation efficiency(P>0.05).The optimal transformation condition was initial A.tumefaciens GV3101 OD600 nm value=0.8,C.pyrenoidosa concentration 107 CFU/ml,coated and mixed(100μl)on the CM tablet with pH 5.4 and acetosyringone concentration 200μmol/L,protected from light at 24℃for 40 h then turn to filter plate,and the transformant was clearly visible only after 2 d,with 88 transformant/106 microalgae count.The overall transformation process took only 5 d,which was more than 3 times shorter than the transformation time of autotrophic microalgae,which provided an applicable technique for the metabolic engineering modification of C.pyrenoidosa,as well as provided theoretical basis for short term efficient genetic transformation of other heterotrophic microalgaes.