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甘蔗梢腐病是世界范围内重要的经济作物真菌性病害,其病原真菌为串珠镰孢菌(F.moniliforme)。通过农杆菌介导的遗传转化构建了串珠镰孢菌(菌株CNO-1)的突变体库,并通过Hi TAIL-PCR和菌落形态对突变体库进行鉴定。结果构建了库容量为956的CNO-1突变体库。从CNO-1突变体库中随机挑选6个转化子,进行PCR鉴定,结果均为阳性。CNO-1突变体库中,筛选出生长速度减慢突变体22个,色素变异突变体1个;对47株突变体进行Hi TAIL-PCR,扩增插入位点侧翼序列,得到有效序列22条,BLAST结果显示,T-DNA的22个插入位点分布在CNO-1的15条基因组scaffold上;生长速度减慢突变体的插入失活基因分别与细胞分裂、DNA修复、△12脂肪酸脱氢酶、E3泛素连接酶等有关。
Sugarcane shoot rot is an important fungal disease of economic crops worldwide, and the pathogenic fungus is F. moniliforme. Mutant pools of Fusarium moniliforme (strain CNO-1) were constructed by agrobacterium-mediated genetic transformation and mutant pools were identified by Hi TAIL-PCR and colony morphology. Results A library of CNO-1 mutants with a 956-ku capacity was constructed. Six transformants were randomly selected from the CNO-1 mutant pool for PCR identification and the results were positive. CNO-1 mutant library, 22 mutants with slow growth rate and 1 pigment mutant were screened out. 47 mutants were amplified by Hi TAIL-PCR and the flanking sequences of insertion sites were amplified to obtain 22 effective sequences , BLAST results showed that 22 insertion sites of T-DNA were distributed on 15 genomic scaffolds of CNO-1; the growth-slowing mutants inserted inactivation genes were associated with cell division, DNA repair, △ 12 fatty acid dehydrogenation Enzyme, E3 ubiquitin ligase and so on.