日本落叶松瞬时转化体系的优化及初步应用

Optimization and Application of Transient Transformation System of Larix kaempferi

[目的]优化农杆菌介导的日本落叶松胚性愈伤组织瞬时转化体系。[方法]以液体增殖培养7 d的日本落叶松胚性愈伤组织为受体材料,利用携带β-葡糖醛酸酶基因(GUS)的pCAMBIA1305.1载体进行瞬时转化,根据GUS的表达量及酶活性,筛选最佳侵染液浓度、侵染时间和共培养时间。并利用筛选出的转化体系,分析落叶松scarecrow-like 6(LaSCL6)启动子的活性。[结果]瞬时转化后,GUS表达明显。当侵染液浓度OD600为0.2,侵染5 min,共培养72 h时,GUS的表达量最高,△CT值为-2.274 2;当侵染液浓度OD600为0.05,侵染5 min,共培养72 h时,GUS酶活性最高,为25.728 6 U·L^(-1)。LaSCL6启动子的活性是CaMV35S启动子的1.55倍。[结论]综合考虑GUS的表达量和酶活性,当侵染液浓度OD600为0.05,侵染5 min,共培养24 h时,GUS的表达量和酶活性较高,这一条件可以用来进行日本落叶松胚性愈伤组织的高效转化。

[Objective]To optimize an Agrobacterium-mediated transient transformation system with Larix kaempferi embryogenic callus.[Methods]The embryogenic callus of Larix kaempferi cultured in liquid me-dium for 7 days was used as the receptor material,and pCAMBIA1305.1 vector carrying β-glucuronidase(GUS)was used for transient transformation.Based on the expression level and enzyme activity of GUS,the optimal infection solution concentration,infection time and co-culture time were screened.The activity of Larix kaempferi scarecrow-like 6(LaSCL6)promoter was analyzed with the screened transformation system.[Results]After transient transformation,the expression of GUS was obvious.When the concentra-tion of infection solution was 0.2,the infection lasted for 5 minutes,and the co-culture time was 72 hours,GUS expression was the highest,with-2.2742.When the concentration of infection solution was 0.05,the infection lasted for 5 minutes,and the co-culture time was 72 hours,GUS enzyme activity was the highest with 25.7286 U/L.The activity of LaSCL6 promoter was 1.55 times higher than that of CaMV35S pro-moter[Conclusion]In view of the expression level and enzyme activity of GUS,transformation efficiency is high when the concentration of infection solution is 0.05,the infection time is 5 minutes,and the co-culture time is 24 hours,which can be used for efficient transformation of embryogenic callus of Larix kaempferi.