Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (2): 279-294.doi: 10.16420/j.issn.0513-353x.2021-1081
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ZHANG Xin, QI Yanxiang, ZENG Fanyun, WANG Yanwei, XIE Peilan, XIE Yixian*(), PENG Jun*(
)
Received:
2022-06-16
Revised:
2022-09-27
Online:
2023-02-25
Published:
2023-03-06
Contact:
*(E-mail:CLC Number:
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.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2021-1081
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 |
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 |
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.
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.
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).
Fig. 8 Phenotypic analysis of hpRNAi-FCC1 induced gene silencing in ΔFocDCL1 and ΔFocDCL2 ΔFocFCC1 with red colony phenotypes served as positive control.
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 |
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 |
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.
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