摘要：

在建立T-DNA插入突变体库的基础上,为筛选出致病力明显下降的突变体并明确其插入位点信息,对突变体的培养表型和致病性差异进行测试,研究结果为进一步阐明尖孢镰刀菌致病分子机制奠定基础。在前期构建的T-DNA插入突变体库中,采用PCR检测筛选了300株T-DNA插入突变体,观察和测定菌落形态、生长速率、产孢量、遗传稳定性等生物学性状,用海棠胚根接种法比较突变体致病力差异,对筛选到的致病力明显减弱的突变体进行插入位点侧翼序列分析及Southern杂交分析。结果显示,283个供试突变体都已插入目的基因,并伴有绿色荧光。将T-DNA插入突变体连续转接培养5代后,在含潮霉素B的培养基上能稳定遗传,且菌落形态和颜色无明显改变;在对283株突变体进行致病性测定中,87.9%的突变菌株致病力减弱,在致病力减弱的突变体中,HS2-520、HS2-1016和HS2-2109几乎丧失了致病性。选取8株致病力明显减弱的突变体进行Southern杂交分析,结果显示,其中6株突变体为单拷贝插入菌株,1株为双拷贝插入菌株。对这8株突变体进行了TAIL-PCR侧翼序列扩增,经公司测序,与野生菌株HS2基因组序列比对后获得12条侧翼序列,明确了其中3条侧翼序列为尖孢镰刀菌序列。获得致病力明显减弱的单拷贝插入突变体6株,明确了其中2株突变体的插入位点侧翼序列信息。

关键词: 尖孢镰刀菌, T-DNA插入突变体, Southern杂交, TAIL-PCR, 致病力

Abstract:

Based on the establishment of the T-DNA insertion mutant library,in order to screen out the mutants with significantly decreased pathogenicity and clarify the insertion site information,the phenotypic and pathogenicity differences of the mutants were tested.The results laid a foundation for further elucidating the pathogenic molecular mechanism of Fusarium oxysporum.In our previous work,a T-DNA insertion library of F.oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation(ATMT).In the present study,totally 300 transformants generated by T-DNA insertions were randomly selected and the alien gene was detected via PCR.The colony morphology,growth rate,sporulation,mitosis stability and other biological traits were observed and measured. The pathogenicity differences of mutants were compared by begonia radicle inoculation method. The flanking sequence analysis of insertion site and Southern Blot analysis were performed on the mutants with significantly weakened pathogenicity.The results showed that 283 tested mutants had inserted the target gene with green fluorescence.After the T-DNA insertion mutant was continuously transferred for five generations,it could be stably inherited on the medium containing hygromycin B,and the colony morphology and color had no significant change.In the pathogenicity test of 283 mutants,the pathogenicity of 87.9% mutant strains was weakened.Among the attenuated mutants,HS2-520,HS2-1016 and HS2-2109 almost lost pathogenicity.Eight mutants with significantly weakened pathogenicity were selected for Southern Blot analysis.The results showed that six mutants were single-copy inserted strains and one was double-copy inserted strains.The TAIL-PCR flanking sequences of the eight mutants were amplified.After sequencing by the company,12 flanking sequences were obtained after comparison with the genomic sequence of wild strain HS2.And three of them were identified as gene sequences of F.oxysporum.Six single copy insertion mutants with significantly reduced pathogenicity were obtained,and flanking sequence information of insertion sites of two mutants were confirmed.

Key words: Fusarium oxysporum, T-DNA insertion mutant, Southern hybridization, TAIL-PCR, Pathogenicity