农杆菌介导的棉花枯萎病菌转化体系的优化

Optimization of Agrobacterium tumefaciens-mediated transformation system for Fusarium oxysporum

摘要:

[背景] 海岛棉相对陆地棉更易感枯萎病，一旦发生很难根治，使得枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害，但其致病机理目前还不是十分明确。[目的] 揭示棉花枯萎病菌的遗传变异和致病机理，同时获得带有绿色荧光蛋白（Green Fluorescent Protein，GFP）标记的棉花枯萎病菌转化子用于观察其侵染海岛棉的途径。[方法] 采用农杆菌介导的遗传转化（Agrobacteriumtumefaciens-Mediated Transformation，ATMT）方法，对棉花枯萎病菌7号生理小种st89进行了遗传转化并对转化条件进行优化。[结果] 农杆菌介导的遗传转化法转化棉花枯萎病菌的最佳条件为：150 mg/L的潮霉素浓度能完全抑制棉花枯萎病菌的生长，浓度为200 mg/L的头孢噻肟钠能完全抑制农杆菌LBA4404生长，农杆菌起始浓度OD₆₀₀为0.2，农杆菌预培养时间为8 h，棉花枯萎病菌分生孢子浓度为10⁵个/mL，枯萎病菌孢子悬液和农杆菌LBA4404比例为1：1，乙酰丁香酮浓度为200 μmol/mL，共培养时间为4 d，转化后培养温度25℃。利用优化的转化系统将GFP基因转入到棉花枯萎病菌中，转化效率最高可以达到252±7.37个转化子/10⁵个孢子。PCR扩增以及荧光观察表明GFP基因能够正常表达。[结论] 转GFP基因的枯萎病菌的获得为深入研究棉花枯萎病入侵的机理奠定了基础。

Abstract:

[Background] Sea island cotton is more susceptible to fusarium wilt than upland cotton. Once it occurs, it is difficult to cure, making fusarium wilt gradually become the main disease threatening the development of sea island cotton industry in Xinjiang, but the pathogenesis is still unclear. [Objective] In order to reveal the genetic variation and pathogenic mechanism of Fusarium oxysporum, the green fluorescent protein (GFP) labeled transformants of F. oxysporum were obtained and used to observe its infection pathway to sea island cotton. [Methods] Agrobacterium tumefaciens-mediated transformation, Agrobacterium tumefaciens-mediated transformation (ATMT) mediated genetic transformation of cotton fusarium wilt race st89 is carried out and the transformation conditions were optimized. [Results] The optimum conditions for ATMT transformation of F. oxysporum:150 mg/L hygromycin could completely inhibit the growth of F. oxysporum, 200 mg/L cefotaxime sodium completely inhibited the growth of Agrobacterium LBA4404, the initial concentration of Agrobacterium is OD₆₀₀ 0.2, the pre-culture time of Agrobacterium is 8 h, The concentration of conidia is 10⁵ spores/mL, the volume ratio of spore suspension of F. oxysporum to Agrobacterium LBA4404 is 1:1. The concentration of acetosyringone in co-culture is 200 μmol/mL, co-culture time is 4 d, culture temperature after transformation 25℃. Using the optimized transformation system, GFP gene is successfully transferred into F. oxysporum. Maximum transformation efficiency can be achieved 252±7.37 transformants/10⁵ spores. PCR amplification and fluorescence observation showed that GFP gene could be expressed normally. [Conclusion] The acquisition of GFP transgenic F. oxysporum laid a foundation for further study on the invasion mechanism of cotton Fusarium wilt.