曾凡云 王艳玮 漆艳香 谢艺贤 张欣 彭军

摘  要：本研究克隆鑒定了香蕉枯萎病菌4号生理小种（Foc4）的细胞周期蛋白C1（Fusarium Cyclin C1，FCC1）基因FocFCC1，采用Split-marker同源重组技术获得基因敲除突变体ΔFocFCC1，通过比较突变体和野生菌株在生长速度、产孢量和致病力等方面的差异，探索了FocFCC1基因缺失对香蕉枯萎病菌生物学功能的影响，并评估了FocFCC1作为枯萎菌内源报告基因的可行性。结果表明：与Foc4野生型菌株相比，ΔFocFCC1菌丝生长缓慢、菌丝畸形及产孢量减少，对巴西蕉苗的致病力明显减弱，推测FocFCC1基因在Foc4的生长发育、产孢及致病力等方面具有重要作用。以FocFCC1为靶标基因，评估了两种基因敲除重组方法介导的基因敲除效率，利用红色表型作为判断依据，挑取再生板上有红色表型的转化子进行PCR检测，Split-marker重组方法获得阳性转化子比例为91.7%，而多片段装配融合方法获得阳性转化子的比例为64%。ΔFocFCC1出现典型红色菌落，红色表型与PCR检测的FocFCC1阳性敲除转化子一一对应，FocFCC1基因敲除后红色表型肉眼容易分辨，可作为香蕉枯萎病菌内源报告基因探索新的遗传操作技术在该菌上应用的可行性。

关键词：香蕉枯萎菌;细胞周期蛋白C1;基因敲除;表型分析;致病力

中图分类号：S668.1      文献标识码：A

Abstract： In this study， the Fusarium Cyclin C1 gene FocFCC1 in F. oxysporum f. sp. cubense race 4 （Foc4） was cloned and identified. The ΔFocFCC1 gene-knockout mutants were obtained by the Split-marker homologous recombination technique. The growth rate， sporulation， pathogenicity were studied to investigate the biological function of FocFCC1 in Foc4. Additionally， we evaluated whether the FocFCC1 could serve as an endogenous reporter gene applied in Foc4. The results demonstrated that ΔFocFCC1 showed slow growth rate， hyphal deformity， decreased conidium production， and significantly reduced pathogenicity to banana （Cavendish， AAA）. Therefore， we hypothesized that FocFCC1 gene might play an important role in the growth， sporulation and pathogenicity of Foc4. Further， a comparative study of the efficiency of two different homologous DNA disruption construct methods was carried out， one is Split-marker recom-bination approach， another termed Multi-fragment assembly by In-fusion cloning， requires only one standard PCR reaction and one fragment assembly reaction. Using the red phenotype as the judging basis， the transformants with red phenotype on the regeneration plate were selected for PCR detection. The results showed the percentage of positive transformants of Split-marker recombination approach was 91.7%， while that of the Multi-fragment assembly by In-Fusion cloning method was 64%. Next， ΔFocFCC1 showed typical red colony distinguished with Foc4， and the red colony phenotypes coupled with the positive PCR amplification bands. It is important that positive transformants can pick up according to the red phenotype by eyes without PCR procedures， and can serve as endogenous reporter gene for further Foc4 molecular biology research.

Keywords： Fusarium. oxysporum f. sp. cubense; Fusarium Cyclin C1; gene knockout; phenotype analysis; patho-genicity test

DOI： 10.3969/j.issn.1000-2561.2021.11.011

香蕉枯萎病（Fusarium oxysporum f. sp. cu-bense，Foc）是破坏香蕉维管束导致植株死亡的毁灭性土传病害，是香蕉产业上最关注的焦点问题之一，也是热带地区植物病理学研究的热点之一。随着真菌遗传操作技术的发展和成熟，利用遗传操作技术研究香蕉枯萎病菌的基因功能也日趋成熟，但是相对模式病原菌而言，香蕉枯萎病菌的研究中很多新的分子生物学技术还没有被完全建立并应用，因此迫切需要鉴定内源报告基因并以此评估新技术应用的可行性。……