农杆菌介导的高卢蜜环菌遗传转化体系的优化

Optimization of Agrobacterium-Mediated Genetic Transformation System of Armillaria gallica

目的:该研究在已有高卢蜜环菌遗传转化体系的基础上,对遗传转化体系进行优化以提高转化效率,为后续蜜环菌分子标记辅助育种、基因功能的研究奠定基础。方法:首先构建遗传转化的质粒pH101-PAgGPD-GFP-TrpC,将其转化到大肠埃希菌中扩大培养并提取质粒。提取的质粒分别转化LBA4404,EHA105,GV3101,AGL-1共4种不同的农杆菌。用转化后的农杆菌浸染高卢蜜环菌,筛选出转化率最高的农杆菌。再分别从抗生素种类及浓度、共培养时间、菌液浓度和浸染方法 5个方面对农杆菌介导的高卢蜜环菌遗传转化体系进行优化。采用Synbiosis ProtoCol 3测量影像分析仪观察蜜环菌在不同条件下的表型谱。结果:优化后高卢蜜环菌遗传转化条件为农杆菌菌株采用EHA105,菌液浓度为吸光度A_(600nm)=0.6;共培养时间为2 d,浸染方式为负压浸染10 min,初筛培养基为含400 mg·L^(-1)头孢噻肟钠,10 mg·L^(-1)潮霉素的PDA培养基,复筛培养基为含12 mg·L^(-1)潮霉素的PDA培养基。结论:该研究在已经建立的高卢蜜环菌的遗传转化体系的基础上成功对该体系进行了优化,且优化前后的转化率差异存在明显统计学意义(P<0.05),即优化后高卢蜜环菌的转化效率约为4.33%,相比于优化前提高了约8倍。

Objective: To optimize the existing genetic transformation system of Armillaria gallica to improve the transformation efficiency and lay a foundation for the follow-up research on Armillaria molecular marker-assisted breeding and gene function. Method: The genetically transformed plasmid pH101-PAgGPDGFP-TrpC was constructed,transformed into Escherichia coli,amplified,and cultured,and the plasmid was extracted. The extracted plasmid was transformed into four different agrobacteria LBA4404,EHA105,GV3101,and AGL-1,respectively. The transformed agrobacteria were used for impregnating A. gallica,and the agrobacteria with the highest conversion rate were screened out. Then the agrobacterium-mediated genetic transformation system of A. gallica was optimized from the type and concentration of antibiotics,co-culture time,concentration of bacterial solution, and impregnation method. The phenotype profiles of A. gallica under different conditions were observed using Synbiosis ProtoCol 3. Result: The optimized genetic transformation conditions of A. gallica were as follows:the Agrobacterium strain of EHA105 at absorbance A_(600nm)=0.6,the co-culture time of 2 d,the infection mode of negative pressure impregnation for 10 min,the primary screening medium of PDA medium containing 400 mg·L^(-1) cefotaxime sodium and 10 mg·L^(-1) hygromycin,and the secondary screening medium of PDA medium containing 12 mg·L^(-1) hygromycin. Conclusion: In this study,the existing genetic transformation system of A. gallica was optimized,and there was a significant difference in the transformation rate before and after optimization(P<0.05). After optimization,the transformation efficiency of A. gallica was about 4.33%,which was about eight times higher than that before optimization.