Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (8): 216-224.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1457
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ZHAO Jing-ya(), PENG Meng-ya, ZHANG Shi-yu, SHAN Yi-xuan, XING Xiao-ping, SHI Yan, LI Hai-yang, YANG Xue, LI Hong-lian, CHEN Lin-lin()
Received:
2021-11-23
Online:
2022-08-26
Published:
2022-09-14
Contact:
CHEN Lin-lin
E-mail:zhaojy0108@163.com;llchensky@163.com
ZHAO Jing-ya, PENG Meng-ya, ZHANG Shi-yu, SHAN Yi-xuan, XING Xiao-ping, SHI Yan, LI Hai-yang, YANG Xue, LI Hong-lian, CHEN Lin-lin. Role of C2H2 Zinc Finger Transcription Factor FpCzf7 in the Growth and Pathogenicity of Fusarium pseudograminearum[J]. Biotechnology Bulletin, 2022, 38(8): 216-224.
Primer | Sequence(5'-3') |
---|---|
F1 | CATCAAATGCAGGAACGGAGC |
R1 | CAATATCATCTTCTGTCGACGGTTTCATGGGCTGC- TAGAAATC |
F2 | ATAGAGTAGATGCCGACCGCGGGTTCCATCGTCT- TGCCGTGGTGAATTG |
R2 | CAATTGAACGAGGAAACACG |
HYG/F[ | GTCGACAGAAGATGATATTG |
HYG/R[ | GAACCCGCGGTCGGCATCTACTCTAT |
YG/F[ | GATGTAGGAGGGCGTGGATATGTCCT |
HY/R[ | GTATTGACCGATTCCTTGCGGTCCGAA |
F3 | GACCTACCCATGTTTACTTG |
R3 | CTTCGAAATCTGCATCGCATG |
G1 | CAACATCATATCGAACCCAC |
G2 | GAGAGTGAGAAGACATGG |
H850F[ | TTCCTCCCTTTATTTCAGATTCAA |
H852R[ | ATGTTGGCGACCTCGTATTGG |
H855R[ | GCTGATCTGACCAGTTGC |
H856F[ | GTCGATGCGACGCAATCGT |
PKNTG-F | CTATAGGGCGAATTGGGTACCGACCTACCCATGTT- TACTTG |
PKNTG-R | GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCT- GCTCTTG |
nei-F | GACCACGGTTTCCACTACTC |
GFP-R | GATGCCCTTCAGCTCGATGCGGTTCA |
RTF | TTCAACACCACCAGCATCA |
RTR | TGTGGTGCAAGTTCTCGTT |
TEF1a-RTF | TCACCACTGAAGTCAAGTCC |
TEF1a-RTR | ACCAGCGACGTTACCACGTC |
Table 1 Primers used in this study
Primer | Sequence(5'-3') |
---|---|
F1 | CATCAAATGCAGGAACGGAGC |
R1 | CAATATCATCTTCTGTCGACGGTTTCATGGGCTGC- TAGAAATC |
F2 | ATAGAGTAGATGCCGACCGCGGGTTCCATCGTCT- TGCCGTGGTGAATTG |
R2 | CAATTGAACGAGGAAACACG |
HYG/F[ | GTCGACAGAAGATGATATTG |
HYG/R[ | GAACCCGCGGTCGGCATCTACTCTAT |
YG/F[ | GATGTAGGAGGGCGTGGATATGTCCT |
HY/R[ | GTATTGACCGATTCCTTGCGGTCCGAA |
F3 | GACCTACCCATGTTTACTTG |
R3 | CTTCGAAATCTGCATCGCATG |
G1 | CAACATCATATCGAACCCAC |
G2 | GAGAGTGAGAAGACATGG |
H850F[ | TTCCTCCCTTTATTTCAGATTCAA |
H852R[ | ATGTTGGCGACCTCGTATTGG |
H855R[ | GCTGATCTGACCAGTTGC |
H856F[ | GTCGATGCGACGCAATCGT |
PKNTG-F | CTATAGGGCGAATTGGGTACCGACCTACCCATGTT- TACTTG |
PKNTG-R | GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCT- GCTCTTG |
nei-F | GACCACGGTTTCCACTACTC |
GFP-R | GATGCCCTTCAGCTCGATGCGGTTCA |
RTF | TTCAACACCACCAGCATCA |
RTR | TGTGGTGCAAGTTCTCGTT |
TEF1a-RTF | TCACCACTGAAGTCAAGTCC |
TEF1a-RTR | ACCAGCGACGTTACCACGTC |
Fig. 1 Domains and expression levels of FpCzf7 in F. pseudograminearum A:The C2H2 zinc finger motifs in FpCzf7. B:The relative expression levels of FpCzf7 during infection stages. MY and IF18h,IF30h,IF48h,IF3d,IF5d,IF7d represent the wild-type mycelia and the F. pseudograminearum infected the wheat coleoptile at 18 h,30 h,48 h,3 d,5 d,and 7 d,respectively
Fig. 2 FpCzf7 gene knockout in F. pseudograminearum A:Schematic representation of the FpCzf7 deletion strategy and PCR amplification(M:DNA size marker;A1:a 964 bp fragment of FpCzf7 upstream;A2:a 997 -bp fragment of FpCzf7 downstream;HPH:a 1 350 bp fragment of the hygromycin phosphotransferase gene;A1H1:a 1 762 bp fragment of FpCzf7 and HPH upstream cassette;H2A2:a 1 928 bp fragment of FpCzf7 and HPH downstream cassette). B:PCR assay of the FpCzf7 deletion mutants(M:DNA size marker;H:a 750 bp fragment of the HPH;G:a 751 bp fragment of the FpCzf7 gene;F:a 1 673 bp fragment of FpCzf7 and HPH upstream cassette;R:a 1 568 bp fragment of FpCzf7 and HPH downstream cassette)
Fig. 3 Mycelia growth and morphology assays of WT,Δfpczf7 and Δfpczf7-cp on PDA plates A:Colonies morphology for 3 d. B:Colony diameters for 3 d(**P < 0.01,t-test). C:Hyphal morphology for 24 h(Bar=50 µm)
Fig. 4 Conidia production and germination assays A:Conidia and germinated tubes of WT,Δfpczf7 and Δfpczf7-cp. B:Production of conidia of WT,Δfpczf7 and Δfpczf7-cp(**P < 0.01,t-test). C:Conidia germination rates of WT,Δfpczf7 and Δfpczf7-cp
Fig. 5 Inoculation on barley leaves and wheat coleoptiles A:Barley leaves inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. B:The lesion diameters on barley leaves(**P < 0.01,t-test). C:Wheat coleoptiles were inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. D:Lesion lengths on wheat coleoptiles(**P < 0.01,t-test)
Fig. 6 Experiments of pot-culture and wheat heads inoculation A:Wheat seedlings inoculated by WT,Δfpczf7 and Δfpczf7-cp for 10 d. B:The disease severity index of Fusarium crown rot.(**P < 0.01,t-test). C:Wheat heads were inoculated with conidial suspension of WT,Δfpczf7 and Δfpczf7-cp strains for 20 d
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