通过缺失大丽轮枝菌Vdku80构建其高效基因敲除受体菌株

doi:10.3864/j.issn.0578-1752.2014.11.008

摘要/Abstract

摘要： 【目的】验证大丽轮枝菌（Verticillium dahliae）非同源末端连接途径关键基因Vdku80的功能，构建高效的大丽轮枝菌基因敲除受体菌株。【方法】利用常规基因敲除的方法，构建大丽轮枝菌Vdku80基因缺失的突变体菌株ΔVdku80。即将Vdku80的同源重组DNA片段（Vdku80的基因组上下游DNA片段之间连接潮霉素抗性基因）通过农杆菌介导的转化转入大丽轮枝菌，通过同源区段的重组，潮霉素抗性基因便会替换掉野生型Vdku80，从而获得突变体菌株ΔVdku80。对突变体菌株ΔVdku80的生物学表型，即生长速率、产孢量和对棉花的致病性进行测试。分别以大丽轮枝菌野生型菌株和ΔVdku80作为受体菌株，采用上述基因敲除方法测试2个几丁质合成酶编码基因ChsV和ChsVI的基因敲除效率。【结果】成功构建了Vdku80缺失突变体ΔVdku80。野生型和突变体菌株ΔVdku80在PDA培养基上的菌落形态相似，且培养8 d后菌落直径无明显差别。野生型和突变体菌株ΔVdku80产孢高峰均出现在摇瓶培养后第5天，且该时期产孢量亦无明显的变化。浓度为0.02%的MMS对野生型和突变体菌株ΔVdku80的生长均有明显的抑制作用，且抑制程度相同。接种野生型和突变体菌株ΔVdku80 4周后，棉株真叶均开始表现出萎蔫、褪绿等发病症状，野生型和突变体菌株ΔVdku80对棉花造成的病情指数分别为54.2±4.9和53.6±5.4，亦无明显变化。因此，突变体菌株ΔVdku80的生长速率、产孢量和致病性相对于野生型菌株均未发生明显变化。使用野生型菌株作为基因敲除的受体菌株，几丁质合成酶编码基因ChsV和ChsVI的基因敲除转化子在总转化子中的比例分别为44%和31%；而使用ΔVdku80作为基因敲除的受体菌株，几丁质合成酶编码基因ChsV和ChsVI基因敲除转化子在总转化子中的比例分别为94%和87%。【结论】Vdku80缺失的菌株ΔVdku80作为受体菌株可以提高其他基因的基因敲除效率，且Vdku80的缺失对大丽轮枝菌的生长速率、产孢量和致病性均无影响，因此不会干扰其他基因的敲除所带来的表型变化。Vdku80缺失的菌株ΔVdku80可作为高效的大丽轮枝菌基因敲除受体菌株。

关键词: 大丽轮枝菌 , 基因敲除效率 , 同源重组 , 非同源末端连接

Abstract: 【Objective】 The objective of this study is to characterize Vdku80 of Verticillium dahliae, which encodes the protein involved in the nonhomologous end-joining double DNA break repair pathway, and to provide a recipient strain with enhanced gene knockout frequency. 【Method】 V. dahliae Vdku80 deletion mutant ΔVdku80 was constructed as previously reported. Briefly, Vdku80 gene knockout was accomplished through a combination of techniques, beginning in Vdku80 homologous DNA fragment construction. Individual V. dahliae cells were genetically transformed via Agrobacterium tumefaciens mediated transformation. Recombination then occurred in the region of that sequence within the gene of Vdku80, resulting in the insertion of hygromycin resistance gene to disrupt the gene of Vdku80. Vegetable growth, sporulation and pathogenicity of ΔVdku80 were studied compared with those of wild type strain. The effect of the absence of Vdku80 on gene knockout frequency was tested by disruption of ChsV and ChsVI, which encoded V. dahliae chitin synthases.【Result】Vdku80 deletion mutant strain (ΔVdku80) was successfully constructed. Deletion of Vdku80 had no effect on colony morphology and mycelial growth, as deduced from the monitoring of radial growth of wild type strain and ΔVdku80 on PDA media 8 days after inoculation. Both wild type strain and ΔVdku80 reached highest level of conidia production after growing for 5 days on Czapek media and showed no significant differences with respect to conidia number in this moment. Growth of wild-type strain and ΔVdku80 was inhibited by 0.02% MMS, and the inhibition rate was also similar. Finally, the Vdku80 deletion mutant ΔVdku80 was qualitatively and quantitatively as pathogenic as its corresponding wild-type strains. Altogether, these results showed that the deletion of Vdku80 did not affect mycelial growth, conidia production and pathogenicity of V. dahliae. Using wild-type strain as a gene knockout recipient, gene knockout frequency was 44% and 31% for ChsV and ChsVI, respectively, while using ΔVdku80 as gene knockout recipient, gene knockout frequency was 94% and 87% for ChsV and ChsVI, respectively.【Conclusion】Vdku80 deletion mutant ΔVdku80 displayed wild-type phenotypes regarding growth, sporulation and pathogenicity, but clearly improved gene knockout frequency of other genes. These results support that ΔVdku80 can be a recipient strain with enhanced gene knockout frequency in V. dahliae.

Key words: Verticillium dahliae , gene knockout frequency , homologous recombination , nonhomologous end-joining

田李1, 刘娜1, 徐荣旗2, 曲志才1, 刘乾1. 通过缺失大丽轮枝菌Vdku80构建其高效基因敲除受体菌株[J]. 中国农业科学, 2014, 47(11): 2142-2150.

TIAN Li-1, LIU Na-1, XU Rong-Qi-2, QU Zhi-Cai-1, LIU Qian-1. Construction of Enhanced Gene Knockout Frequency Recipient Strain by Deletion of Vdku80 in Verticillium dahliae[J]. Scientia Agricultura Sinica, 2014, 47(11): 2142-2150.

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