农杆菌介导转化莱茵衣藻体系的优化

Optimization of Agrobacterium-mediated transformation of Chlamydomonas reinhardtiii

摘要:

[目的] 为建立根癌农杆菌介导的莱茵衣藻快速简便高效的遗传转化体系，本研究以模式生物莱茵衣藻为受体材料，从转化方法和转化子快速鉴定两个方面进行了优化。[方法] 比较了固体培养基共培养转化方法和液体培养基共培养转化方法对根癌农杆菌LBA 4404介导的莱茵衣藻CC425转化效率的影响；研究并比较了（1）首先经过TE裂解再进行PCR（两步法）和（2）不经TE裂解直接进行PCR（一步法）的两种转化子鉴定方法的最佳反应条件和扩增效率。[结果] 农杆菌LBA 4404和莱茵衣藻CC425液体培养基共培养5 d后的转化效果最好，转化率达43.33±1.67个转化子/10⁶个藻细胞。PCR最佳反应条件为：使用高保真DNA聚合酶Taq 1进行扩增；参加PCR反应的细胞密度为5×10³-5×10⁶个/mL；TE裂解缓冲液沸水浴20 min（两步直接PCR方法），或者预变性15 min（一步直接PCR方法）。两步法直接PCR的扩增效率优于一步法，但后者反应步骤更简洁。[结论] 本研究建立并优化了农杆菌液体介导莱茵衣藻遗传转化体系，该体系可快速获得遗传转化子，减少转化工作量。

Abstract:

[Objective] To establish a rapid, simple and efficient Agrobacterium-mediated genetic transformation system for Chlamydomonas reinhardtiii, we used the model organism C. reinhardtiii as the receptor material and optimized the Agrobacterium-mediated transformation system of C. reinhardtiii from two aspects: transformation method and transformants identification method. [Methods] We compared the effect of solid co-culture and liquid co-culture on the transformation efficiency of C. reinhardtii CC425 mediated by A. tumefaciens LBA4404. Besides, we analyzed the optimal reaction conditions and amplification efficiency of (1) two-step PCR after TE cleavage, and (2) one-step PCR without TE cleavage. [Results] The highest transformation efficiency was achieved by a 5-day liquid-medium co-culture of Agrobacterium and Chlamydomonas. The transformation rate was 43.33±1.67 transformants/10⁶ algal cells. The optimal reaction conditions were: amplification with high fidelity DNA polymerase Taq 1; the cell density involved in PCR was 5×10³–5×10⁶ cells/mL; before amplification, cells were boiled in TE lysis buffer for 20 min (two-step PCR method), or initial denaturation for 15 min (one-step direct PCR method). The amplification efficiency of two-step PCR is better than that of one-step PCR, but the latter is more concise. [Conclusion] Agrobacterium-mediated transformation system of C. reinhardtii was established and optimized, through which rapid genetic transformation can be fulfilled and the workload could be reduced.