棉花枯萎病菌突变体库的构建及分析

Construction and analysis of mutant library of cotton Fusarium wilt

摘要:

[背景] 棉花枯萎病逐渐成为威胁新疆海岛棉产业发展的主要病害，但关于棉花枯萎病菌的致病力、产孢量、生长速度及颜色变化等相关功能基因目前还不是十分明确。[目的] 通过构建绿色荧光蛋白（green fluorescent protein，GFP）标记棉花枯萎病菌突变体库，筛选出由于T-DNA的随机插入而导致性状发生变异的突变体，为棉花枯萎病菌功能基因筛选和研究提供材料。[方法] 通过Agrobacterium tumefaciens mediated transformation （ATMT）构建了GFP标记的棉花枯萎病菌的突变体库，检测分析T-DNA插入情况及转化子稳定性，对随机选取的突变体菌落形态、生长速率、产孢量、萌发率、T-DNA插入拷贝数及致病力进行分析，从而筛选获得变异明显且稳定的突变体。[结果] 利用优化后的ATMT介导体系转化获得了1 600株GFP标记的棉花枯萎病菌转化子；转化子在不含潮霉素B的PDA培养基上连续转接7代再转到含潮霉素B的培养基上仍能正常生长，说明潮霉素Hyg基因成功插入野生型基因组且稳定遗传。最终筛选出17株菌落表型与野生型有差异的突变体，其中包括生长缓慢型、菌丝深紫色型、菌丝淡紫色型、菌丝浅黄色型；突变体A-1致病力相较于野生型增加了21.98%，A-1产孢量相较于野生型增加了103.54%，C-6、C-7产孢量相较于野生型降低61.90%。[结论] 构建了农杆菌介导GFP标记的棉花枯萎病菌突变体库，筛选分析获得了菌落形态、生长速率、产孢量和致病力发生变化的突变体，为进一步研究棉花枯萎病菌相关功能基因筛选奠定了基础。

Abstract:

[Background] Cotton Fusarium wilt has gradually become a major disease threatening the development of Gossypium barbadense cotton industry, but the related functional genes of cotton Fusarium wilt are not very clear. [Objective] In this study, a mutant library of F. oxysporum marked by green fluorescent protein (GFP) was constructed to screen mutants with random insertion of T-DNA, which could provide materials for the screening and research of functional genes of F. oxysporum. library of F. oxysporum marked by GFP was constructed.[Methods] The mutant library of GFP-labeled F. oxysporum was constructed by Agrobacterium tumefaciens mediated transformation (ATMT), and the T-DNA insertion and transformation stability were detected and analyzed. The colony morphology, growth rate, sporulation, germination rate, T-DNA insertion copy number and pathogenicity of randomly selected mutants were analyzed, so as to screen mutants with obvious variation and stability. [Results] 1 600 GFP-labeled F. oxysporum transformants were obtained by using the optimized ATMT mediated system. The transformants were transferred to PDA medium without hygromycin B for 7 generations and then transferred to the medium with hygromycin B. The transformants could still grow normally, indicating that hygromycin gene Hyg was successfully inserted into the wild-type genome and stably inherited. Finally, 17 mutants with different colony phenotypes were screened, including slow growth type, dark purple mycelium type, light purple mycelium type, and light yellow mycelium type. Compared with the wild type, the pathogenicity of mutant A-1 increased by 21.98%, the sporulation of mutant A-1 increased by 103.54%, and the sporulation of mutant C-6 and C-7 decreased by 61.90%. [Conclusion] The mutant library of cotton Fusarium wilt marked by GFP mediated by Agrobacteriumwas constructed, and the mutants with changes in colony morphology, growth rate, sporulation and pathogenicity were screened and analyzed, which laid a foundation for further study of functional genes related to cotton fusarium wilt.