Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (5): 112-119.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1247
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PAN Guo-qiang1(), WU Si-yuan1,2, LIU Lu1, GUO Hui-ming1, CHENG Hong-mei1, SU Xiao-feng1()
Received:
2022-10-10
Online:
2023-05-26
Published:
2023-06-08
Contact:
SU Xiao-feng
E-mail:15704610584@163.com;suxiaofeng@caas.cn
PAN Guo-qiang, WU Si-yuan, LIU Lu, GUO Hui-ming, CHENG Hong-mei, SU Xiao-feng. Construction and Preliminary Analysis of Verticillim dahliae Mutant Library[J]. Biotechnology Bulletin, 2023, 39(5): 112-119.
引物名称Primer name | 序列 Sequence(5'-3') |
---|---|
VdrDNA-qPCR-F | CCGCCGGTCCATCAGTCTCTCTGTTTATAC |
VdrDNA-qPCR-R | CGCCTGCGGGACTCCGATGCGAGCTGTAAC |
NbActin-qPCR-F | GGCTTCCTCAAGGTCGGCTATG |
NbActin-qPCR-R | GCTGCATGTCATCCCACTTCTTC |
Table 1 Primer sequences for detection of biomass of V. dahliae in Nicotiana benthamiana root by qPCR
引物名称Primer name | 序列 Sequence(5'-3') |
---|---|
VdrDNA-qPCR-F | CCGCCGGTCCATCAGTCTCTCTGTTTATAC |
VdrDNA-qPCR-R | CGCCTGCGGGACTCCGATGCGAGCTGTAAC |
NbActin-qPCR-F | GGCTTCCTCAAGGTCGGCTATG |
NbActin-qPCR-R | GCTGCATGTCATCCCACTTCTTC |
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
AP1 | Provided by Takara Genome Walking Kit, unknown sequence |
SP1 | ATCGTTGGTGTCGATGTCAGCTCC |
SP2 | GCGTTTCGGGTTTACCTCTTCCAG |
SP3 | CGAGATCAAGCAGATCAACGGTCG |
Table 2 Primer sequences for the amplification of flanking sequences at insertion site
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
AP1 | Provided by Takara Genome Walking Kit, unknown sequence |
SP1 | ATCGTTGGTGTCGATGTCAGCTCC |
SP2 | GCGTTTCGGGTTTACCTCTTCCAG |
SP3 | CGAGATCAAGCAGATCAACGGTCG |
Fig. 1 Schematic diagram and PCR amplification of hygromycin resistance gene expression cassette in pUC-Hyg A: Schematic diagram of hygromycin resistance gene expression cassette in pUC-Hyg. B: PCR amplification of hygromycin resistance gene expression cassette fragment. M: DNA marker. 1-4: Hygromycin resistance gene expression cassette fragment. N: Negative control(ddH2O)
Fig. 2 PCR identification of randomly inserted mutants of V. dahlia M: DNA marker. 1-5: PCR identification products of transformant 1-5 genomic DNA. WT: PCR identification products of V991 genomic DNA. N: Negative control(ddH2O)
Fig. 3 Colony growth phenotype of randomly inserted mutants on PDA and different carbon source medium A: Colony morphology of V. dahliae V991 and 5 randomly inserted mutants were cultured on PDA and different carbon source medium for 12 d. B: Colony diameters of V. dahliae V991 and 5 randomly inserted mutants were cultured on PDA and different carbon source medium for 12 d. C: Spore production of V. dahliae V991 and 5 randomly inserted mutants cultured on PDA and different carbon source medium for 12 d. Error bars indicate the standard deviations and different letters indicate significant differences at P<0.05
Fig. 4 Pathogenicity of randomly inserted mutants of V. dahliae to N. benthamiana A: Disease incidence of N. benthamiana plants after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. Mock: N. benthamiana plants that were not inoculated with V. dahliae. B: Disease index of N. benthamiana plants after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. C: Fungal biomass in the root tissues of N. benthamiana after inoculation with V. dahliae V991 and 5 randomly inserted mutants for 12 d. Error bars indicate the standard deviations and different letters indicate significant differences at P<0.05
Fig. 5 Flanking sequence analysis of insertion site for randomly inserted mutant M5 A: Flanking sequence detection of the insertion site of hygromycin resistance gene expression cassette fragment(M: DNA marker. 1-9: three rounds of PCR reaction amplification products). B: Schematic diagram of insertion site of hygromycin resistance gene expression cassette fragment
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