Cloning and Functional Verification of Non-ribosomal Peptide Synthetase Gene Promoter NP249 from Trichoderma longibrachiatum

doi:10.7668/hbnxb.20193783

Abstract

Abstract:

Peptaibols are polypeptide antibacterials synthesized by fungal non-ribosomal peptide synthetases(NRPS),which can inhibit a variety of pathogens,promote plant growth and induce cell apoptosis.In order to further explore the regulatory mechanism of non-ribosomal peptide antimicrobial synthesis in Trichoderma spp.,and to help improve the output of peptaibols through genetic engineering.By designing primers,the upstream promoter region of T.longibrachiatum non-ribosomal peptide synthetase gene NP249 was cloned,and then a green fluorescent protein gene(GFP)fusion expression vector was constructed to verify the promoter of gene NP249.A 1 204 bp activating region of NRPS was amplified by PCR using the primers 249-1B and 249-4X from total DNA of T.longibrachiatum as a template.Replaced the GFP promoter on the vector by promoter NP249.Digestion of pCX-62 vector and promoter fragment with BamH Ⅰ and Xma Ⅰ,respectively,and ligation with T₄ ligase,The GFP fusion vector pCX-62-NP249-GFP was constructed,and the protoplasts of Trichoderma spp..The hydromycin resistant transformer Z249 was screened.The transformer Z249 could detect GFP fluorescence by fluorescence microscopy.The cloned promoter could initiate GFP expression and function as a promoter.

Key words: Trichoderma longibrachiatum, Non-ribosomal peptide synthase, Promoter

GUAN Mengjiao, SUN Xujie, XIA Zhuolin, REN Aizhi, ZHAO Peibao. Cloning and Functional Verification of Non-ribosomal Peptide Synthetase Gene Promoter NP249 from Trichoderma longibrachiatum[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 199-203. doi: 10.7668/hbnxb.20193783.

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Figures/Tables 7

Fig.1 Promoter predictions

Fig.2 PCR amplification and electrophoresis result of NP249 gene fragment after sequence M.DL2000 DNA Marker;1,2.NP249 gene fragment.

Fig.3 Recombinant plasmid electrophoresis results M.DL5000 DNA Marker;1.pMD-18T recombinant plasmid.

Fig.4 PCR electrophoresis result of GFP fusion vector bacterial solution M.DL2000 DNA Marker;1.Target fragment;2.GFP fusion vector bacterial solution PCR.

Fig.5 Result of double enzyme digestion electrophoresis with GFP fusion vector M.DL5000 DNA Marker;1.Double enzyme digestion of GFP fusion vector.

Fig.6 Detection of hph gene in transformants by PCR amplification M.DL2000 DNA Marker;1.DNA of Trichoderma longibrachiatum SMF2;2,3.DNA of transformants.

Fig.7 Fluorescent detection of transformant hypha

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