生物技术进展 ›› 2024, Vol. 14 ›› Issue (3): 422-432.DOI: 10.19586/j.2095-2341.2024.0013
• 研究论文 • 上一篇
席凯飞(
), 李成杰, 丁艺, 郭维()
收稿日期:2024-01-23
接受日期:2024-02-27
出版日期:2024-05-25
发布日期:2024-06-18
通讯作者:
郭维
作者简介:席凯飞 E-mail: kaifeix@163.com；
基金资助:
Kaifei XI(), Chengjie LI, Yi DING, Wei GUO()
Received:2024-01-23
Accepted:2024-02-27
Online:2024-05-25
Published:2024-06-18
Contact:
Wei GUO
摘要：
拟轮枝镰孢菌（Fusarium verticillioides）是引起玉米茎基腐病和穗粒腐病的主要病原菌之一，严重威胁玉米的产量和品质。为了深入研究拟轮枝镰孢菌致病基因的功能，对该菌中非同源末端连接（non-homologous end joining，NHEJ）途径中的2个关键基因FvKu70和FvKu80分别进行了基因敲除以创制高效的基因敲除菌株，并比较了野生型菌株和突变体菌株在营养生长速率、菌落形态、产孢量、对玉米的致病力和基因敲除效率等方面的差异。研究结果表明，FvKu70和FvKu80的基因缺失突变体与野生型FvLNF15-11相比，在PDA平板上的形态特征（如菌丝形态、生长速率、菌落直径、产孢量）没有明显差异，对玉米茎秆的致病力也类似。此外，选择尿嘧啶生物合成相关基因FvpyrG作为敲除的靶基因，分析了FvKu70或FvKu80缺失突变体菌株的同源重组效率，结果显示突变体菌株均显著高于野生型，其中ΔFvKu70的同源重组效率最高。综上所述，FvKu70或FvKu80基因缺失突变体可以快速又高效地实现拟轮枝镰孢菌的基因敲除，为进一步研究该菌的功能基因提供了技术支持。
中图分类号:
席凯飞, 李成杰, 丁艺, 郭维. 利用非同源末端连接缺陷构建拟轮枝镰孢菌的高效基因敲除方法[J]. 生物技术进展, 2024, 14(3): 422-432.
Kaifei XI, Chengjie LI, Yi DING, Wei GUO. Highly Efficient Gene Knockout Method in Fusarium verticillioides Using Nonhomologous End-joining Deficiency[J]. Current Biotechnology, 2024, 14(3): 422-432.
| 引物 | 引物序列（5’→3’） |
|---|---|
| 1F | 5’-GATTACGAATTCGAGCTCGGTACCACTCCTTGTTGGTCTTGCCT-3’ |
| 1R | 5’-ATCTCTAGAGGATCCCCGGGTACCATTGATACGATTGATGTCTG-3’ |
| 2F | 5’-GTCGACCTGCAGGCATGCAAGCTTGACTGAGGTCGAAGGGCAAA-3’ |
| 2R | 5’-AAAACGACGGCCAGTGCCAAGCTTAATATGTAAAAGTCCAACCC-3’ |
| 3F | 5’-GATTACGAATTCGAGCTCGGTACCCTCCGATACCAAGCCAGCA-3’ |
| 3R | 5’-ATCTCTAGAGGATCCCCGGGTACCTCATACCCATCATCGTCTTG-3’ |
| 4F | 5’-GTCGACCTGCAGGCATGCAAGCTTGAGTGAATCATAGGGCCTTG-3’ |
| 4R | 5’-AAAACGACGGCCAGTGCCAAGCTTACATCTCTGGGTGTTGCGAA-3’ |
| 7F | 5’-GGGTAAGGATCACCTTGATA-3’ |
| 7R | 5’-TTAAAGGCAATCGCGATCGA-3’ |
| 8F | 5’-TCCATCAGCATACCACTCCT-3’ |
| 8R | 5’-ATAGACTCGAAGATGGTGAG-3’ |
| HYR | 5’-GTATTGACCGATTCCTTGCGGTCCGAA-3’ |
| YGF | 5’-GATGTAGGAGGGCGTGGATATGTCCT-3’ |
| HYF | 5’-ATGAAAAAGCCTGAACTC-3’ |
| YGR | 5’-TTCCTTTGCCCTCGGACG-3’ |
| Hyg-F | 5’-ATGAAAAAGCCTGAACTCAC-3’ |
| Hyg-R | 5’-CTATTCCTTTGCCCTCGGAC-3’ |
| Ku70F | 5’-GATGACTTGGGTGACATTTC-3’ |
| Ku70R | 5’-GTCCAAAGGAGGAAACTGGT-3’ |
| Ku80F | 5’-GGAGACGAGAAATTCGACCT-3’ |
| Ku80R | 5’-CTGAAGAATTCTGTAGTGCC-3’ |
| pyrG-up-F | 5’-GATTACGAATTCGAGCTCGGTACCCATAAAAACCATGAACCATT-3’ |
| pyrG-up-R | 5’-ATCTCTAGAGGATCCCCGGGTACCGAGGTGAGAGGTGAGAGGTG-3’ |
| pyrG-dw-F | 5’-GTCGACCTGCAGGCATGCAAGCTTTGGAAGTCGGGACGGCCTTG-3’ |
| pyrG-dw-R | 5’-AAAACGACGGCCAGTGCCAAGCTTAGCCTTGTACCTTTCCTTGA-3’ |
| pyrG-u1300-F | 5’-TAACCGTCTCAGACCTGAAAG-3’ |
| pyrG-d1300-R | 5’-TCGGATAGTACTGCCAGTTGA-3’ |
| pyrG-in-F | 5’-CAAGGCCTCGGTTGCATCTC-3’ |
| pyrG-in-R | 5’-CCATGTCATAATGCGTCTTGA-3’ |
| pyrG-F/ | 5’-CGACATTCCACCCATTTACGC-3’ |
| pyrG-R | 5’-GACGTGGTGAATCGGCCGACT-3’ |
表1 本研究所用的引物
Table 1 Primers used in this study
| 引物 | 引物序列（5’→3’） |
|---|---|
| 1F | 5’-GATTACGAATTCGAGCTCGGTACCACTCCTTGTTGGTCTTGCCT-3’ |
| 1R | 5’-ATCTCTAGAGGATCCCCGGGTACCATTGATACGATTGATGTCTG-3’ |
| 2F | 5’-GTCGACCTGCAGGCATGCAAGCTTGACTGAGGTCGAAGGGCAAA-3’ |
| 2R | 5’-AAAACGACGGCCAGTGCCAAGCTTAATATGTAAAAGTCCAACCC-3’ |
| 3F | 5’-GATTACGAATTCGAGCTCGGTACCCTCCGATACCAAGCCAGCA-3’ |
| 3R | 5’-ATCTCTAGAGGATCCCCGGGTACCTCATACCCATCATCGTCTTG-3’ |
| 4F | 5’-GTCGACCTGCAGGCATGCAAGCTTGAGTGAATCATAGGGCCTTG-3’ |
| 4R | 5’-AAAACGACGGCCAGTGCCAAGCTTACATCTCTGGGTGTTGCGAA-3’ |
| 7F | 5’-GGGTAAGGATCACCTTGATA-3’ |
| 7R | 5’-TTAAAGGCAATCGCGATCGA-3’ |
| 8F | 5’-TCCATCAGCATACCACTCCT-3’ |
| 8R | 5’-ATAGACTCGAAGATGGTGAG-3’ |
| HYR | 5’-GTATTGACCGATTCCTTGCGGTCCGAA-3’ |
| YGF | 5’-GATGTAGGAGGGCGTGGATATGTCCT-3’ |
| HYF | 5’-ATGAAAAAGCCTGAACTC-3’ |
| YGR | 5’-TTCCTTTGCCCTCGGACG-3’ |
| Hyg-F | 5’-ATGAAAAAGCCTGAACTCAC-3’ |
| Hyg-R | 5’-CTATTCCTTTGCCCTCGGAC-3’ |
| Ku70F | 5’-GATGACTTGGGTGACATTTC-3’ |
| Ku70R | 5’-GTCCAAAGGAGGAAACTGGT-3’ |
| Ku80F | 5’-GGAGACGAGAAATTCGACCT-3’ |
| Ku80R | 5’-CTGAAGAATTCTGTAGTGCC-3’ |
| pyrG-up-F | 5’-GATTACGAATTCGAGCTCGGTACCCATAAAAACCATGAACCATT-3’ |
| pyrG-up-R | 5’-ATCTCTAGAGGATCCCCGGGTACCGAGGTGAGAGGTGAGAGGTG-3’ |
| pyrG-dw-F | 5’-GTCGACCTGCAGGCATGCAAGCTTTGGAAGTCGGGACGGCCTTG-3’ |
| pyrG-dw-R | 5’-AAAACGACGGCCAGTGCCAAGCTTAGCCTTGTACCTTTCCTTGA-3’ |
| pyrG-u1300-F | 5’-TAACCGTCTCAGACCTGAAAG-3’ |
| pyrG-d1300-R | 5’-TCGGATAGTACTGCCAGTTGA-3’ |
| pyrG-in-F | 5’-CAAGGCCTCGGTTGCATCTC-3’ |
| pyrG-in-R | 5’-CCATGTCATAATGCGTCTTGA-3’ |
| pyrG-F/ | 5’-CGACATTCCACCCATTTACGC-3’ |
| pyrG-R | 5’-GACGTGGTGAATCGGCCGACT-3’ |
图1 FvKu70及其同源蛋白进化树分析注：Fusarium verticilllioides—拟轮枝镰孢菌; Neurospora crassa—粗糙脉孢霉; N. crassa—粗糙脉孢霉; Chaetomium globosum—球毛壳菌; Pyricularia grisea—稻瘟病; Phaeoacremonium minimum—褐枝顶孢霉; Verticillium dahlia—大丽轮枝菌; Lecanicillium saksenae—刀孢蜡蚧菌; Botrytis cinerea—灰霉菌; Sclerotinia sclerotiorum—核盘菌; Macrophomina phaseolina—菜豆壳球孢菌; Parastagonospora nodorum—旁星孢杆菌; Coccidioides immitis—粗球孢子菌; Blastomyces gilchristii—吉氏芽生菌; Aspergillus clavatus—棒曲霉; A. fumigatus—烟曲霉; A. terreus—土曲霉; A. oryzae—米曲霉; A. sojae—酱油曲霉; A. parasiticus—寄生曲霉; Saccharomyces cerevisiae—酿酒酵母。
Fig. 1 The phylogenetic tree of FvKu70 and its homologous proteins
图2 FvKu80及其同源蛋白进化树分析注：Fusarium verticilllioides—拟轮枝镰孢菌; Lecanicillium saksenae—刀孢蜡蚧菌; Verticillium dahliae—大丽轮枝菌; Phaeoacremonium minimum—褐枝顶孢霉; Chaetomium globosum—球毛壳菌; Neurospora crassa—粗糙脉孢霉; N. crassa—粗糙脉孢霉; Pyricularia grisea—稻瘟菌; Botrytis cinerea—灰霉菌; Sclerotinia sclerotiorum—核盘菌; Parastagonospora nodorum—旁星孢杆菌; Macrophomina phaseolina—菜豆壳球孢菌; Coccidioides immitis—粗球孢子菌; Blastomyces gilchristii—吉氏芽生菌; Aspergillus clavatus—棒曲霉; A. fumigatus—烟曲霉; A. terreus—土曲霉; A. oryzae—米曲霉; A. sojae—酱油曲霉; A. parasiticus—寄生曲霉; Saccharomyces cereuisiae—酿酒酵母。
Fig. 2 The phylogenetic tree was constructed to analyze the evolutionary relationships between FvKu80 and its homologous proteins.
图3 FvKu70和FvKu80基因敲除示意图和载体构建A:FvKu70基因敲除示意图；B：FvKu80基因敲除示意图；C：FvKu70的上下游片段L1/R1和FvKu80基因的上下游片段L2/R2；D：扩增用于Split敲除FvKu70时导入原生质体的片段LH1和RH1；E：扩增用于Split敲除FvKu80时导入原生质体的片段LH2和RH2；M—DNA maker
Fig. 3 The gene knockout diagram and construction of knock-out cassette of FvKu70 and FvKu80
图4 ΔFvKu70和ΔFvKu80的PCR及Southern blot鉴定A: ΔFvKu70的PCR检测，1~4分别为利用Ku70F/R、7F/YGR、HYF/7R和HYF/YGR检测野生型菌株基因组中的目标条带，5~8分别为利用Ku70F/R、7F/YGR、HYF/7R和HYF/YGR检测突变体菌株基因组中的目标条带；B：ΔFvKu70的Southern blot验证结果；C：ΔFvKu80的PCR检测，1~4分别为利用Ku80F/R、8F/YGR、HYF/8R和HYF/YGR检测野生型菌株基因组中的目标条带，5~8分别为利用Ku80F/R、8F/YGR、HYF/8R和HYF/YGR检测突变体菌株基因组中的目标条带；D：ΔFvKu80的Southern blot验证结果；M—DNA maker
Fig. 4 PCR and Southern blot identification of ΔFvKu70 and ΔFvKu80
图5 ΔFvKu70和ΔFvKu80基因敲除菌株生长速率测定及菌落形态A： FvLNF15-11、ΔFvKu70和ΔFvKu80在第3天的菌落形态图；B：FvLNF15-11、ΔFvKu70和ΔFvKu80在第5天的菌落直径测量结果；C: FvLNF15-11、ΔFvKu70和ΔFvKu80在第5天的菌落形态图；D：FvLNF15-11、ΔFvKu70和ΔFvKu80培养5 d后的产孢量测定结果
Fig. 5 Assessment of vegetative growth rate and colony morphology in ΔFvKuU70 and ΔFvKu80
图6 ΔFvKu70和ΔFvKu80的致病力测定A: 玉米茎秆分别接种FvLNF15-11、ΔFvKu70和ΔFvKu80 7 d后的症状；B：玉米茎秆分别接种FvLNF15-11、ΔFvKu70和ΔFvKu80 7 d后的病斑面积
Fig. 6 Pathogenicity analysis of ΔFvKu70 and ΔFvKu80 on maize stalks
图7 分别以FvLNF15-11、ΔFvKu70和ΔFvKu80为受体菌敲除FvpyrG的效率评估A: 以FvLNF15-11、ΔFvKu70和ΔFvKu80为受体菌敲除FvpyrG基因的转化平板；B：以FvLNF15-11、ΔFvKu70和ΔFvKu80为受体菌敲除FvpyrG基因的敲除效率；C：以FvLNF15-11、ΔFvKu70和ΔFvKu80为受体菌敲除FvpyrG基因后菌落在添加尿嘧啶和尿嘧啶核苷的MM平板上的生长表型；D：对图C的菌落生长直径测量结果；E：以FvLNF15-11、ΔFvKu70和ΔFvKu80为受体菌敲除FvpyrG基因后菌落在不添加尿嘧啶和尿嘧啶核苷的MM平板上的生长情况
Fig. 7 Evaluation of FvpyrG gene knockout efficiency using FvLNF15-11, ΔFvKu70, and ΔFvKu80 as recipient strains
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