Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (9): 161-170.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0047
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LIU Sha-yu(), CAO Jian, LI Meng, LIU Zhi-qiang, LI Xiao-yu()
Received:
2021-01-11
Online:
2021-09-26
Published:
2021-10-25
Contact:
LI Xiao-yu
E-mail:1091293310@qq.com;hdlixiaoyu@126.com
LIU Sha-yu, CAO Jian, LI Meng, LIU Zhi-qiang, LI Xiao-yu. Biological Function of a Zn2Cys6 Transcription Factor CgAswA in Colletotrichum gloeosporioides[J]. Biotechnology Bulletin, 2021, 37(9): 161-170.
Fig. 1 Homologous recombination principle of the gene CgaswA Sur is the chlorimuron-ethyl resistance gene,and black triangles indicate the primers used to verify gene-knockout mutants
Primer | Primer sequence(5'-3') |
---|---|
CgaswAupF | CGGAATTCTCAGAAGAGGCACCGCAACGCAT |
CgaswAupR | CCCAAGCTTATCTCCTTCATCCGACACGACTG |
CgaswAF | ATGAACGACAACATACTCTACC |
CgaswAR | TCAAGTGCACAAGAAGTCGAGG |
CgaswAdownF | GCTCTAGACGCTCAACCACCTCATCGCTACC |
CgaswAdownR | CGGAATTCCCGTCCGTTGTCTTGACCTTTGC |
CgaswAUU | CTCCTTCGCTCACTTGCTCGCTC |
PI | CAGGGTTTTCCCAGTCACGACGTTG |
PI1 | GTATGTTGTGTGGAATTGTGAGCGG |
CgaswADD | GCACAATGGAGATCTTCGCCCTT |
CgaswAhbF | CGGAATTCTGCTTTCGCTCCCTCTGTTTCTT |
CgaswAhbR | GCTCTAGAGTCCGTTGTCTTGACCTTTGCTG |
Table 1 Primer and sequence
Primer | Primer sequence(5'-3') |
---|---|
CgaswAupF | CGGAATTCTCAGAAGAGGCACCGCAACGCAT |
CgaswAupR | CCCAAGCTTATCTCCTTCATCCGACACGACTG |
CgaswAF | ATGAACGACAACATACTCTACC |
CgaswAR | TCAAGTGCACAAGAAGTCGAGG |
CgaswAdownF | GCTCTAGACGCTCAACCACCTCATCGCTACC |
CgaswAdownR | CGGAATTCCCGTCCGTTGTCTTGACCTTTGC |
CgaswAUU | CTCCTTCGCTCACTTGCTCGCTC |
PI | CAGGGTTTTCCCAGTCACGACGTTG |
PI1 | GTATGTTGTGTGGAATTGTGAGCGG |
CgaswADD | GCACAATGGAGATCTTCGCCCTT |
CgaswAhbF | CGGAATTCTGCTTTCGCTCCCTCTGTTTCTT |
CgaswAhbR | GCTCTAGAGTCCGTTGTCTTGACCTTTGCTG |
Fig. 2 Protein domain and phylogenetic tree of CgAswA protein A:Protein domain analysis of CgAswA. B:Phylogenetic analysis of CgAswA. The sequence ID is in parentheses;the numbers on the horizontal lines represent evolutionary distances
Fig. 3 Verification of CgaswA gene knockout and com-plementation A:PCR results of CgaswAF / CgaswAR;B:PCR results of CgaswAUU / PI;C:PCR results of PI1 / CgaswADD. M:DL2 000 DNA marker;1:wild type;2:ΔCgaswA-3;3:ΔCgaswA-8;4:ΔCgaswA-11;5:ΔCgaswA/aswA
Fig. 4 Comparison of strains’ growth on four media A:Growth of strains on four media. B:Statistical analysis of colony diameter. a,b:Significant level,P<0.05
Fig. 5 Sporulation,germination and appressorium formation of wild type,ΔCgaswA and ΔCgaswA/aswA A:Statistical results of conidia yield. B:Statistical results of conidia germination rate. C:Statistical results of appressorium formation rate. D:Conidial germination and appressorium formation at 4 h,8 h and 12 h;co:conidium,gt:germ tube,ap:appressorium;a,b,c,d:significant level,P<0.05. Scale bars = 20 µm
Fig. 6 Onion epidermis infection and cellophane penetration assays A:Appressorium infection assay of different strains on onion epidermis. B:Cellophane penetration assay of conidia suspensions. C:Cellophane penetration assay of mycelial plugs;co:conidium,ap:appressorium,ih:invasive hyphae;a,b,c,d:significant level,P<0.05;scale bars = 20 µm
Fig. 7 Pathogenicity analyses of wild type,ΔCgaswA and ΔCgaswA/aswA A:Symptoms on rubber leaves at 4 d post-inoculation using mycelial plugs and statistical analyses of the diameters of disease spots. B:Symptomss on rubber leaves at 5 d post-inoculation using conidial suspensions and statistical analyses of the diameters of disease spots. 1:CK;2:WT;3:ΔCgaswA/ aswA;4:ΔCgaswA-3;5:ΔCgaswA-8;6:ΔCgaswA-11;a,b,c,d:significant level,P<0.05
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