Cloning and Functional Identification of Trichothecene Mycotoxin Biosynthesis Gene Promoter from Paramyrothecium roridum

doi:10.13560/j.cnki.biotech.bull.1985.2020-1386

Abstract

Abstract:

The promoters of trichothecene mycotoxin biosynthetic genes tri5 and tri12 from endophytic fungus Paramyrothecium roridum were cloned by genome walking,and the core regions of the promoters were identified using prokaryotic,eukaryotic and luciferase expression systems,respectively. The results showed that 12-f1,the promoter fragment of tri12,was of the highest transcriptional efficiency for the expression of ampicillin resistance genes and hygromycin resistance genes;meanwhile,5-f0,the promoter fragment of tri5,demonstrated the highest transcriptional efficiency for luciferase gene expression. Analysis of the functional components of the promoters revealed that tri5 contained TATA box and CAAT box,while tri12 was a TATA-less promoter,but contained CAAT box and GC box. This study lays a foundation for the screening of strong promoters and efficient biosynthesis of trichothecenes.

Key words: Paramyrothecium roridum, trichothecene mycotoxin, promoter, cloning, identification

CEN You-fei, ZHU Mu-zi, YE Wei, LI Sai-ni, ZHONG Guo-hua, ZHANG Wei-min. Cloning and Functional Identification of Trichothecene Mycotoxin Biosynthesis Gene Promoter from Paramyrothecium roridum[J]. Biotechnology Bulletin, 2021, 37(8): 85-94.

Figures/Tables 9

Table 1 Specific reverse primer sequences of the target genes tri5 and tri12

目的基因Target gene	反向引物Reverse primer sp1（5'-3'）	反向引物Reverse primer sp2（5'-3'）	反向引物Reverse primer sp3（5'-3'）
tri5	TGTACTTGACGTTCTCCAGCAAC	GGGAGATTCTGAGAGAGATAGATA	TCTGTCTGTCTGATCTGTCTGTC
tri12	GCGTTGGTCAACATGAAGAATGG	ACATCTGCGGCGATGTTTGAGAT	ATCAAACGAGGGCTCCGATAGTA

Fig.1 Target fragments of tri5 and tri12 obtained by chromosome walking M：Marker trans2K plus;A：The PCR amplification products of tri5 after three rounds of chromosome walking;B：The PCR amplification products of tri12 after three rounds of chromosome walking

Table 2 Core sequences of tri5 and tri12 promoters

目的基因启动子 Promoter of target gene	分值 Score	核心序列 Core sequence
tri5	0.71	ACCAGAGACGGGAATCACGCGCATT- TCTGGCTACGATATCCCCCCTAACG
tri12	0.98	AGGCCCTCGATAAGAAGCGGCGGCG- GGGCCTCGACCAGGAATCGACTACC

Fig.3 Colony PCR verification of DH5α and promoter vector maps M：Marker trans2K plus. A：Colony PCR of DH5α-pET30a-5-f0-Amp. B：Vector map of pET30a-5-f0-Amp. C：Colony PCR of DH5α-YEp352-12-f2-HYRB-CYC1. D：Vector map of YEp352-12-f2-HYRB-CYC1

Fig.4 Screening of E. coli containing different promoter fragments on resistance plates with different concentrations of ampicillin A：The result of culture on plates of DH5α-pET30a-5-f0/5-f1/5-f2/12-f0/12-f1/12-f2-Amp. B：The result of culture on plates of DH5α-pET30a-5-f0/5-f1/5-f2-Amp. C：OD value bar graph of DH5α-YEp352-12-f1-HYRB-CYC1 and DH5α-pET30a-12-f1-Amp. N：DH5α-pET30a-ACP empty carrier（Negative control）;P：DH5α-YEp352-12-f1-HYRB-CYC1（Positive control）;5（f0-f2）：DH5α-pET30a-5-f0/5-f1/ 5-f2-Amp;12（f0-f2）：DH5α-pET30a-12-f0/12-f1/12-f2-Amp

Fig.5 Screening of S. cerevisiae containing different prom-oter fragments on resistant plates with different concentrations of hygromycin A：The result of culture on plates of BJ5464-YEp352-TEF1-HYRB-CYC1（Positive control）,BJ5464-YEp352-No pro-HYRB-CYC1（Negative control）,BJ5464-YEp352-12-f1-HYRB-CYC1 and BJ5464-YEp352-5-f0-HYRB-CYC1;B：OD value bar graph of BJ5464-YEp352-TEF1-HYRB-CYC1（Positive control）and BJ5464-YEp352-12-f1-HYRB-CYC1. N：BJ5464-YEp352-No pro-HYRB-CYC1;P：BJ5464-YEp352-TEF1-HYRB-CYC1;5-f0：BJ5464-YEp352-5-f0-HYRB-CYC1;12-f1：BJ5464-YEp352-12-f1-HYRB-CYC1

Fig.6 Luciferase activity detection of pGL3-5-f0-basic,pGL3-12-f1-basic and pGL3-pgpd-basic vectors in DH5α

Fig.7 Functional component prediction of gene tri5 and tri12

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