香蕉枯萎病菌Dicer-like基因的功能分析

doi:10.16420/j.issn.0513-353x.2021-1081

园艺学报 ›› 2023, Vol. 50 ›› Issue (2): 279-294.doi: 10.16420/j.issn.0513-353x.2021-1081

香蕉枯萎病菌Dicer-like基因的功能分析

张欣, 漆艳香, 曾凡云, 王艳玮, 谢培兰, 谢艺贤^*(), 彭军^*()

中国热带农业科学院环境与植物保护研究所，农业农村部热带作物有害生物综合治理重点实验室，海南省热带农业有害生物监测与控制重点实验室，海口 571101

收稿日期:2022-06-16 修回日期:2022-09-27 出版日期:2023-02-25 发布日期:2023-03-06
通讯作者: *（E-mail：yixian81@126.com，swaupj2@126.com）
基金资助:
海南省自然科学基金项目(322RC756);中国热带农业科学院环境与植物保护研究所自主选题项目(hzsjy2019004);海南省基础与应用基础研究计划（自然科学领域）高层次人才项目(2019RC269);中国热带农业科学院基本科研业务费专项资金项目(1630042018010);国家现代农业产业技术体系建设专项资金项目(CARS-31-07)

Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

ZHANG Xin, QI Yanxiang, ZENG Fanyun, WANG Yanwei, XIE Peilan, XIE Yixian^*(), PENG Jun^*()

Key Laboratory of Integrated Pest Management on Tropical Crops，Ministry of Agriculture and Rural Affairs，Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests，Environment and Plant Protection Institute，Chinese Academy of Tropical Agricultural Sciences，Haikou 571101，China

Received:2022-06-16 Revised:2022-09-27 Online:2023-02-25 Published:2023-03-06
Contact: *（E-mail：yixian81@126.com，swaupj2@126.com）

摘要/Abstract

摘要：

香蕉枯萎病病原菌尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense）4号生理小种（Foc4）含有两个进化上高度保守的Dicer-like基因FocDCL1和FocDCL2。为了探究该基因的功能及其在RNAi中的作用机制，利用同源重组的方法获得ΔFocDCL1、ΔFocDCL2和ΔFocDCL1/2基因敲除突变体。表型分析显示，与Foc4野生型相比，突变体菌丝的营养生长无显著差异，但产孢量下降；ΔFocDCL2突变体在非生物胁迫刚果红处理后菌落明显变小且气生菌丝增多；ΔFocDCL2和ΔFocDCL1/2的致病力下降。miRNA深度测序数据显示，与Foc4野生型相比，ΔFocDCL1、ΔFocDCL2和ΔFocDCL1/2敲除突变体的miRNA长度分布和5′-端首位碱基出现频率都发生了改变，都能产生自身特异miRNA。DCL功能存在交叉和冗余，miRNA可以通过依赖独立的、交叉的或者联合的DCL发生；此外，鉴定到不依赖DCL形成的miRNA。这些结果表明FocDCL在产孢量、非生物胁迫、致病力以及小RNA发生中发挥作用。

关键词: 香蕉, 枯萎病, Fusarium oxysporum f. sp. cubense, Dicer-like基因, 致病力测试, miRNA

Abstract:

The fungal pathogen Fusarium oxysporum f. sp. cubense（Foc）is an important soil-borne pathogenic fungus which causes Banana Fusarium wilt. The Foc race 4（Foc 4）attacking almost all Cavendish cultivars is currently noteworthy in global banana production. In order to explore the function of DCL-like genes in Foc4，the gene-knockout mutants ΔFocDCL1，ΔFocDCL2 and ΔFocDCL1/2 were obtained by homologous recombination technique. The phenotype analysis showed the mutants showed no significant differences on vegetative growth but significant reduced in conidia production compared to the Foc4. The ΔFocDCL2 mutant increased sensitive to Congo red，and the mutant significantly reduced the size of the colony and increased the number of aerial hyphae after Congo red treatment. Next，the pathogenicity test showed that virulence of ΔFocDCL2 and ΔFocDCL1/2 were significantly reduced compared with Foc4，respectively. Furthermore，miRNA deep sequencing data showed the length distribution of the total reads，and the frequency of the 5′ first nucleotide bias of the mutants were markedly changed compared with Foc4，respectively. The miRNA could be generated depending on individual，alternative or joint DCL manners，indicating the FocDCL with overlapped and redundant function in small RNA biogenesis. In addition，DCLs-independent miRNA also be identified in the sequencing data. These results indicated that FocDCL function involved in the stress response，conidia，pathogenicity and generation of miRNAs in Foc4.

Key words: banana, Fusarium wilt, Fusarium oxysporum f. sp. cubense, Dicer-like gene, pathogenicity test, miRNA

中图分类号:

S668.1

张欣, 漆艳香, 曾凡云, 王艳玮, 谢培兰, 谢艺贤, 彭军. 香蕉枯萎病菌Dicer-like基因的功能分析[J]. 园艺学报, 2023, 50(2): 279-294.

ZHANG Xin, QI Yanxiang, ZENG Fanyun, WANG Yanwei, XIE Peilan, XIE Yixian, PENG Jun. Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4[J]. Acta Horticulturae Sinica, 2023, 50(2): 279-294.

图/表 13

表1 FocDCL单敲除和双敲除引物

Table 1 The FocDCLs single and double gene knock-out primers used in this study

引物 Primer	序列（5′-3′） Sequence	引物 Primer	序列（5′-3′） Sequence
HYG-F	CTTGGCTGGAGCTAGTGGAGGT	NEO-F	TCTAGATTAACGCTTACAATTTCC
HYG-R DCL1-LBCK	CCCGGTCGGCATCTACTCTATTC CCAGGCTATGGTCCCAAGAA	NEO-R DCL2-NEO-LBCK	TCAGAAGAACTCGTCAAGAAGG CAGCAGATGTAATAGTCGCCG
DCL1-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGCAAGAGTCCGCTACAATCTCAA	DCL2-NEO--LB-R	GAAATTGTAAGCGTTAATCTAGCCTTGATGCCCTCCTTATCC
DCL1-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGAGCGTTAGAAGCGTAGACAA	DCL2-NEO-RB-F	CCTTCTTGACGAGTTCTTCTGATTGAGAGTGCGGAGGGACTG
DCL1-RBCK	CAACAAACAAGACCTCCTCTC	DCL2-NEO-RBCK	GAGGGTGAGATGAACGGTGA
DCL1-LB-F	GCAAAGAGTCTATCGTGTGAGCC	DCL2-NEO-LB-F	CGACTTACACAAATACATCCTCCC
HYG-R1	GGATGCCTCCGCTCGAAGTA	NEO-R1	GAGCAAGGTGAGATGACAGGAG
HYG-F1	CGTTGCAAGACCTGCCTGAA	NEO-F1	CACCACTCGATCCGTCACCAAC
DCL1-RB-R	CGGTAAAGGATTGGGATTGTTG	DCL2-NEO-RB-R	CGTCTTTGTCTCCATCAACTTCG
HPT-LBCK	GACAGACGTCGCGGTGAGTT	NEO-LBCK	GAATGTCGTCAAGCGGGAAC
HPT-RBCK	TCTGGACCGATGGCTGTGTAG	NEO-RBCK	CGACCACCAAGCGAAACATC
DCL1-1784F	GTCTCTCTCTTTCTGCTGACCG	pFCC1-F1-XhoI	AATTCTCGAGAATTGATACGGCTGGCGAAG
DCL1-3031R	TCAAGGCTGGGATTCAACTTAC	pFCC1-R1-HindⅢ	AGAAAGCTTTCTCAACACCAAGGCCAGT
DCL2-LBCK	AGCATTCGTCAACTTTGCCA	pFCC1-F2-BglII	GGCAGATCTTCTCAACACCAAGGCCAGT
DCL2-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGTCCATCAGCACTCACATCACTC	pFCC1-R2-KpnI FCC1-qRT-F	CCAGGTACCAATTGATACGGCTGGCGAAG TCGACAGCAACGTGGAGATT
DCL2-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGGCATCACTAAACACTCCTCCTTGT	FCC1-qRT-R DCL1-qRT-F	ACCTGTTGATCTGTTCGCGA AGAACAAGTCCTGGCTCTCC
DCL2-RBCK	GAGGGTGAGATGAACGGTGAC	DCL1-qRT-R	GTCGCAATCTGGAACGTCGA
DCL2-LB-F	ACGCTTGGAGAGAATGCGAG	DCL2-qRT-F	TCGATGGAGTTGTGGAGTCA
DCL2-RB-R	AGCCATCAGTCGTAAGAGCAA	DCL2-qRT-R	GCATTCTCCGCAGCTTTGGT
DCL2-988F	CACCTCATTCGCTCACTCTACG	qFocUBI-1F	CCAACCCTGACGATCCTCTTGTGC
DCL2-2373R	ATCACAACCCGACTTCCAGC	qFocUBI-1R	TACTTTCGAGTCCACTCCCGAGCTG

表1 FocDCL单敲除和双敲除引物

Table 1 The FocDCLs single and double gene knock-out primers used in this study

引物 Primer	序列（5′-3′） Sequence	引物 Primer	序列（5′-3′） Sequence
HYG-F	CTTGGCTGGAGCTAGTGGAGGT	NEO-F	TCTAGATTAACGCTTACAATTTCC
HYG-R DCL1-LBCK	CCCGGTCGGCATCTACTCTATTC CCAGGCTATGGTCCCAAGAA	NEO-R DCL2-NEO-LBCK	TCAGAAGAACTCGTCAAGAAGG CAGCAGATGTAATAGTCGCCG
DCL1-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGCAAGAGTCCGCTACAATCTCAA	DCL2-NEO--LB-R	GAAATTGTAAGCGTTAATCTAGCCTTGATGCCCTCCTTATCC
DCL1-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGAGCGTTAGAAGCGTAGACAA	DCL2-NEO-RB-F	CCTTCTTGACGAGTTCTTCTGATTGAGAGTGCGGAGGGACTG
DCL1-RBCK	CAACAAACAAGACCTCCTCTC	DCL2-NEO-RBCK	GAGGGTGAGATGAACGGTGA
DCL1-LB-F	GCAAAGAGTCTATCGTGTGAGCC	DCL2-NEO-LB-F	CGACTTACACAAATACATCCTCCC
HYG-R1	GGATGCCTCCGCTCGAAGTA	NEO-R1	GAGCAAGGTGAGATGACAGGAG
HYG-F1	CGTTGCAAGACCTGCCTGAA	NEO-F1	CACCACTCGATCCGTCACCAAC
DCL1-RB-R	CGGTAAAGGATTGGGATTGTTG	DCL2-NEO-RB-R	CGTCTTTGTCTCCATCAACTTCG
HPT-LBCK	GACAGACGTCGCGGTGAGTT	NEO-LBCK	GAATGTCGTCAAGCGGGAAC
HPT-RBCK	TCTGGACCGATGGCTGTGTAG	NEO-RBCK	CGACCACCAAGCGAAACATC
DCL1-1784F	GTCTCTCTCTTTCTGCTGACCG	pFCC1-F1-XhoI	AATTCTCGAGAATTGATACGGCTGGCGAAG
DCL1-3031R	TCAAGGCTGGGATTCAACTTAC	pFCC1-R1-HindⅢ	AGAAAGCTTTCTCAACACCAAGGCCAGT
DCL2-LBCK	AGCATTCGTCAACTTTGCCA	pFCC1-F2-BglII	GGCAGATCTTCTCAACACCAAGGCCAGT
DCL2-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGTCCATCAGCACTCACATCACTC	pFCC1-R2-KpnI FCC1-qRT-F	CCAGGTACCAATTGATACGGCTGGCGAAG TCGACAGCAACGTGGAGATT
DCL2-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGGCATCACTAAACACTCCTCCTTGT	FCC1-qRT-R DCL1-qRT-F	ACCTGTTGATCTGTTCGCGA AGAACAAGTCCTGGCTCTCC
DCL2-RBCK	GAGGGTGAGATGAACGGTGAC	DCL1-qRT-R	GTCGCAATCTGGAACGTCGA
DCL2-LB-F	ACGCTTGGAGAGAATGCGAG	DCL2-qRT-F	TCGATGGAGTTGTGGAGTCA
DCL2-RB-R	AGCCATCAGTCGTAAGAGCAA	DCL2-qRT-R	GCATTCTCCGCAGCTTTGGT
DCL2-988F	CACCTCATTCGCTCACTCTACG	qFocUBI-1F	CCAACCCTGACGATCCTCTTGTGC
DCL2-2373R	ATCACAACCCGACTTCCAGC	qFocUBI-1R	TACTTTCGAGTCCACTCCCGAGCTG

图1 FocDCL1（左）和FocDCL2（右）敲除示意图及其突变体的PCR A、F：单基因敲除示意图，箭头指示的为引物所在的位置，HY和YG分别为HYG抗性基因前半部分和后半部分序列；B、G：突变体的上游Up和下游Down重组片段PCR扩增；C、H：突变体的内源基因PCR扩增；D、I：潮霉素B抗性基因PCR扩增；E、J：qRT-PCR检测DCL1和DCL2表达。

Fig. 1 FocDCL1（left）and FocDLC2（right）gene knockout and PCR screening the positive mutants A，F：The schematic map of single-gene knockout，the arrows indicate the position of the primers，HY and YG indicate the first and second part of HYG resistance gene，respectively；B，G：Amplification of up and down recombinant from mutants by PCR；C，H：Amplification of endogenous gene by PCR；D，I：Amplification of Hygromycin B resistance gene by PCR；E，J：qRT-PCR detection of DCL1 and DCL2.

图2 ΔFocDCL1/2双敲除示意图及其突变体基因的检测 A：双敲除示意图。箭头指示的为引物位置，NE和EO分别为NEO抗性基因前半部分和后半部分序列；B：HYG和NEO的PCR扩增。C：FocDCL1和FocDCL2的PCR扩增；D：基因上、下游重组片段PCR扩增；E：qRT-PCR检测DCL1和DCL2表达。

Fig. 2 The schematic diagram of ΔFocDCL1/2 double knockout and the detection of its mutant gene A：The schematic diagram of ΔFocDCL1/2 double knockout. The arrows indicate the position of the primers，Ne and EO indicate the first and second part of NEO resistance gene，respectively；B：Amplification of HYG and NEO by PCR；C：Amplification of FocDCL1 and FocDCL2 by PCR；D：Amplification of up and down recombinant from mutants by PCR；E：qRT-PCR detection of DCL1 and DCL2.

图3 FocDCL的蛋白保守结构域及系统进化树 FocDCL的系统进化树采用邻接法及1 000次重复构建。

Fig. 3 The phylogenetic tree and conserved domain of FocDCL Neighbor-joining method was used with 1 000 bootstrap replicates.

图4 FocDCL敲除突变体的表型不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 4 The phenotype of FocDCL gene knockout mutants Different lowercase letters indicate significant differences（P < 0.05）. The same below.

图5 FocDCL敲除突变体在含非生物胁迫试剂的MM培养基上的菌落

Fig. 5 Colony morphology of FocDCL knockout mutants on MM medium containing abiotic stress reagents

图6 FocDCL敲除突变体在含非生物胁迫试剂的MM培养基上的菌落生长抑制率

Fig. 6 Growth inhibition rate of FocDCL knockout mutants on MM medium containing abiotic stress reagents

图7 香蕉幼苗接种FocDCL敲除突变体菌株后叶片和球茎发病症状括号内数字为病情指数。不同小写字母表示差异显著（P < 0.05）。

Fig. 7 Symptoms of banana leaves and corms after inoculation with FocDCLs knockout mutants Disease index in parentheses. Different lowercase letters indicate significant difference（P < 0.05）.

图8 hpRNAi-FCC1诱导ΔFocDCL1和ΔFocDCL2突变体表型分析紫红色的ΔFocFCC1突变体作为正对照。

Fig. 8 Phenotypic analysis of hpRNAi-FCC1 induced gene silencing in ΔFocDCL1 and ΔFocDCL2 ΔFocFCC1 with red colony phenotypes served as positive control.

图9 hpRNAi-FCC1沉默转化子中FCC1的相对表达量

Fig. 9 The relative mRNA expression level of FCC1 in silencing transformant

表2 小RNA测序及DCL敲除突变体miRNA数据统计

Table 2 The statistics of small RNA sequence date and miRNAs of DCL knockout mutants

样品 Sample ID	总数量 Total reads	匹配数量 Mapped reads	已知miRNA Known-miRNA	新的miRNA Novel-miRNA	靶标基因 Target gene
Foc4	18 931 444	6 494 076	16	39	3 829
ΔFocDCL1	8 922 109	2 851 863	10	33	1 772
ΔFocDCL2	9 046 433	3 373 316	9	14	1 105
ΔFocDCL1/2	19 831 731	11 454 808	6	11	818

图10 FocDCL敲除突变体小RNA序列统计 A：unisRNA的长度分布。B：小RNA的5′末端碱基偏向性分析。C：特异miRNA统计。

Fig. 10 Statistics analysis of small RNA sequence from FocDCL knockout mutants A：The length distribution of unique reads of sRNA. B：Nucleotide frequency of the 5′ end of small RNAs；C：Statistics of specific miRNA.

图11 FocDCL敲除突变体与Foc4野生型miRNA比对（A）及miRNA的FocDCL依赖方式鉴定（B）

Fig. 11 The miRNA comparison between FocDCL knockout mutants and Foc4（A）and identification of FocDCL-dependent miRNAs（B）

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香蕉枯萎病菌Dicer-like基因的功能分析

Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

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香蕉枯萎病菌Dicer-like基因的功能分析

Functional Analysis of Dicer-like Genes in Fusarium oxysporum f. sp. cubense Race 4

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