农杆菌介导的携带egfp基因载体转化寡雄腐霉

Transformation of enhanced green fluorescent protein gene in Pythium oligandrum mediated by Agrobacterium tumefaciens

摘要:

[目的]寡雄腐霉（Pythium oligandrum Drechsler）是一种对动、植物和环境无害，兼具杀菌和增产效果的生防真菌。通过研究建立农杆菌介导的寡雄腐霉遗传转化体系。[方法]选用EHA105、AGL-1、LBA4404三种农杆菌菌株对寡雄腐霉进行遗传转化研究，通过对影响遗传转化效果的条件参数试验优化，确立适宜寡雄腐霉遗传转化的农杆菌菌株及转化条件，建立农杆菌介导的寡雄腐霉遗传转化体系。[结果]经研究发现，所选3种农杆菌菌株中EHA105菌株对寡雄腐霉的遗传转化效果最好，其次是AGL-1菌株，LBA4404菌株转化效果不好。EHA105菌株经IM（含300 μmol/L AS）诱导培养至OD₆₀₀=0.6时，与浓度为10⁶-10⁷个/mL的寡雄腐霉孢子悬浮液以1-10：1的比例混合，在25-26℃以液体振荡的方式避光共培养72 h（pH 5.0，含300 μmol/L AS），寡雄腐霉菌体液体振荡恢复培养24 h，涂布抗性选择平板筛选寡雄腐霉转化子，即可得到寡雄腐霉基因工程菌株，其转化率可达到130个转化子/10⁶个孢子。[结论]本研究首次构建了农杆菌介导的寡雄腐霉遗传转化体系，研究结果可为寡雄腐霉的生防机制及分子育种研究提供技术支撑。

关键词:寡雄腐霉;生防真菌;农杆菌;遗传转化;egfp基因

Abstract:

[Objective] Pythium oligandrum is an environmental friendly mycoparasitic biocontrol fungus that can antagonize several plant pathogens and promote plant growth. We constructed Agrobacterium tumefaciens-mediated transformation of P. oligandrum.[Methods] Three A. tumefaciens strains:EH105, AGL-1 and LBA4404 were compared to find out the strain which showed the best genetic transformation efficiency within P. oligandrum. The factors of transformation system were screened and optimized to establish the genetic transformation system within P. oligandrum mediated by Agrobacterium tumefaciens.[Results] EHA105 strain showed the best genetic transform efficiency within P. oligandrum among three tested strains, followed by AGL-1 strain, and LBA4404 strain performed poorly. Then, the optimized genetic transformation system was as follows:A. tumefaciens EHA105 (OD₆₀₀=0.6) suspension was mixed with P. oligandrum (10⁶-10⁷ spores per milliliter) suspension in a proportion of 1-10:1, then incubated in dark conditions for 72 h between 25 and 26℃ in the presence of induction medium (pH 5.0) containing acetosyringone at 300 μmol/L. Afterwards, antibiotic was added in to eliminate A. tumefaciens before 24 hours reviving cultivation. In the end, the transformants were selected by medium containing 200 mg/L of Basta and 250 mg/L of cefotaxime sodium on PDA. In this work, egfp was successfully transformed into P. oligandrum with the efficiency of 130 transformants per 10⁶ spores.[Conclusion] We developed an Agrobacterium tumefaciens-mediated genetic transformation system for P. oligandrum for the first time to provide powerful tool for the study of P. oligandrum molecular breeding and biological control mechanisms.

Key words:Pythium oligandrum;biocontrol fungus;Agrobacterium tumefaciens;genetic transformation;enhanced green fluorescent protein gene