Knockout and Function Analysis of FolSid1 Gene in Fusarium oxysporum f.sp.lini

doi:10.7668/hbnxb.20194757

Abstract

Abstract:

The aim was to study the biological function of FolSid1 gene in Fusarium oxysporum f.sp.lini and its protein localization in Fusarium by cloning the gene.The gene sequence of FolSid1 was cloned by homologous comparison with F.oxysporum, and based on the principle of homologous recombination, a gene deletion box containing hydromycin resistance gene(hph)was constructed by Split Marker strategy,and the gene deletion mutant(ΔFolSid1)was obtained by PEG-mediated transfer into protoplasts of the wild type.pZESH1,a green fluorescent expression vector containing FolSid1 gene was constructed,and the subcellular localization of FolSid1-EGFP fusion protein was performed.The results showed that the sequence of FolSid1 gene consisted of 5 392 bp,which contained 3 introns.Compared with the wild type and the external insertion mutant, the knockout mutant ΔFolSid1 conidia showed a significant decrease in yield, although they did not differ in morphology and size; morphological observations revealed that the growth rate of colonies from the knockout mutant was significantly slower. The experiments of subcellular localization showed that FolSid1 protein was located in the cell membrane of mycelia cell.FolSid1 gene regulated the vegetative growth of mycelium,conidiogenesis and pathogenicity of Fusarium oxysporum f.sp.lini.

Key words: Fusarium oxysporum f.sp.lini, FolSid1, Split Marker, Gene disruption

GAO Fang, HOU Zhanming. Knockout and Function Analysis of FolSid1 Gene in Fusarium oxysporum f.sp.lini[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 189-198. doi: 10.7668/hbnxb.20194757.

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http://www.hbnxb.net/EN/Y2025/V40/I1/189

Figures/Tables 12

Tab.1 Primer sequence

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')
FolSid1 1F	CTTGTTGCGTTGCATGATC
FolSid1 2R	CAGCTTCGTGGAGTCGTC
FolSid1 3F	CCGGTACCTTGCCTTAGTG
FolSid1 4R	CAGTTTCGCCAGTTGCC
FolSid1 5F	CGTTGCTGAGCATTATCAGG
FolSid1 6R	GAAGTCCTTGCCGAAGTTG
FolSid1 7F	GCCAACTTCAGCGAAAC
FolSid1 8R	GGAGTATCAGCATGACGG
FolSid1 9F	CGAGCTGCATGCTCAAAC
FolSid1 10R	GGAACGCCGATATGTG
FolSid1 11R	GACAAGATATCGCCGTGG
FolSid1 12R	GAAGAGAACAACGCCTACAAG
FolSid1 KO1F	GGCGTGATATGTGATGTGATATG
FolSid1 KO2R	TTGACCTCCACTAGCTCCAGCCAAGCCG GTAGTCCTGACCGTTGTCTGG
FolSid1 KO3F	ATAGAGTAGATGCCGACCGCGGGTTCC GTGCACAAGGCGTTAAAGG
FolSid1 KO4R	CGATACGCGGTACGCCGAG
HYG-F	GGCTTGGCTGGAGCTAGTGGAGGTCAA
HY-R	TTCGGACCGCAAGGAATCGGTCAATAC
YG-F	GATGTAGGAGGGCGTGGATATGTCCT
HYG-R	ATAGAGTAGATGCCGACCGCGGGTTC
FolSid1 N1F	CGTTGCTGAGCATTATCAGGTCC
FolSid1 N2R	CGCATAGTCGATCCAGAGTTGCC
FolSid1 C1F	CCAGACAACGGTCAGGACTACC
FolSid1 C2R	GACGATGGCTTCCGGTGCTGC
FolSid1 G1F	GCCTGCAGGTCGACTCTAGAGGATCCGC CAACCATCGACGATCGAACC
FolSid1 G2R	CAGCTCCTCGCCCTTGCTCACCATAACG GCAGGCCATCGAAGGCGAAGAC
Neo F	GAGAGGCTATTCGGCTATGACT
Neo R	GGCCACAGTCGATGAATCCAGA
FolSid1-F	GACCATGACTCCTAAGGACGAGC
EGFP-R	TGAAGCACTGCACGCCGT

Fig.1 Amino acid sequence alignment of four fungi

Fig.2 Phylogenetic tree based on amino acid sequences of 10 fungi

Fig.3 Construction of gene deletion box of FolSid1 M.Marker Ⅲ.A.PCR amplification product of upstream and downstream sequence of FolSid1(1.PCR amplification product of upstream sequence of FolSid1; 2.PCR amplification product of downstream sequence of FolSid1).B.PCR amplification product of upstream and downstream fragments of hph;(1.PCR amplification product of upstream fragment of hph; 2.PCR amplification product of downstream fragment of hph).C.Overlapping PCR product for FolSid1 Split Marker;(1.FolSid1 1F/HY-R overlapping PCR product; 2.YG-F/FolSid1 4R overlapping PCR product).

Fig.4 Screening and identification of FolSid1 transformants A.ΔFolSid1 genomic DNA(M.1 kb plus DNA ladder; 1—5.Transformants 1—5 genomic DNA);B.Negative screening of FolSid1 knockout mutants using FolSid1 N1F/FolSid1 N2R as primers; C.Positive screening of FolSid1 knockout mutants using FolSid1 KO1F/HY-R as primers;D.Positive screening of FolSid1 knockout mutants using HYG-F/HYG-R as primers; E.Positive screening of FolSid1 knockout mutants using YG-F/FolSid1 KO4R as primers.B-E:M.Marker Ⅲ;1-4.FolSid1 transformant DNA was used as template;5.hm DNA was used as a template.

Fig.5 Phenotypic analysis of different strains A.Colony morphology of wild type hm,ΔFolSid1and ecFolSid1 cultured on V8 solid medium; B.The mycelium growth rate of hm,ΔFolSid1and ecFolSid1; C.Hyphal morphology of hm,ΔFolSid1and ecFolSid1 in YEPD fluit medium; D.Conidial production of hm,ΔFolSid1and ecFolSid1 in CMC fluit medium.

Tab.2 Conidial production in different strains

菌株 Strain	分生孢子量/(×10⁶/mL) Conidia quantity
hm	7.545±2.339 8a
ΔFolSid1	3.572±0.721 9b
ecFolSid1	6.628±1.060 0a

Fig.6 Infection of flax seedlings by conidia suspension of different strains A.Positive view of infection of different strains conidia suspensions on flax seedlings; B.Top view of infection of different strains conidia suspensions on flax seedlings.

Fig.7 Construction of plasmid pZESH1 A.pZEH1 plasmid DNA; B.Linearized vector pZEH1; C.The PCR amplification product using FolSid1 genomic DNA as template and FolSid1 G1F/FolSid1 G2R as primer.M.1 kb plus DNA ladder.

Fig.8 Screening of plasmid pZESH1 A.The PCR amplification product using different strains as template and FolSid1 N1F/FolSid1 N2R as primer; B.Colony 12,19 plasmid DNA; C.Xba Ⅰ enzyme digestion recombinant plasmid pZESH1; D.The PCR amplification product using plasmid DNA as template and FolSid1 N1F/FolSid1 N2R as primer; E.The PCR amplification product using plasmid DNA as template and FolSid1-F/EGFP-R as primer.M.1 kb plus DNA ladder(A—C);Marker Ⅲ(D—E).

Fig.9 Screening of subcellular locatable transformants A.Transformant DNA of subcellular localization; B.The PCR amplification product using plasmid DNA as template and Neo F/Neo R as primer; C.The PCR amplification product using plasmid DNA as template and FolSid1-F/EGFP-R as primer.M.1 kb plus DNA ladder(A); Marker Ⅲ(B—C).

Fig.10 Subcellular localization of FolSid1-EGFP protein A.FolSid1-egfp strain,arrows indicate the location of mycelium cell membrane; B.hm strain.

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