Paramyrothecium roridum中单端孢霉烯毒素生物合成基因启动子的克隆和功能鉴定

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

生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 85-94.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1386

Paramyrothecium roridum中单端孢霉烯毒素生物合成基因启动子的克隆和功能鉴定

岑由飞¹^,²(), 朱牧孜², 叶伟², 李赛妮², 钟国华¹(), 章卫民²()

1.华南农业大学农业农村部华南作物有害生物综合防治重点实验室天然农药与化学生物学教育部重点实验室,广州510642
2.广东省微生物研究所广东省科学院华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室广东省微生物应用新技术公共实验室,广州 510070

收稿日期:2020-11-14 出版日期:2021-08-26 发布日期:2021-09-10
作者简介:岑由飞,女,硕士研究生,研究方向：微生物功能基因;E-mail： 297288514@qq.com
基金资助:
国家自然科学基金项目(31800063);广东省自然科学基金项目(2019A1515011702);广东省自然科学基金项目(2019A1515011829);广东省科学院百名青年人才培养专项(2020GDASYL-20200104012)

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

CEN You-fei¹^,²(), ZHU Mu-zi², YE Wei², LI Sai-ni², ZHONG Guo-hua¹(), ZHANG Wei-min²()

1. Ministry of Education Key Laboratory of Natural Pesticide and Chemical Biology,Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Integrated Pest Management in South China,South China Agricultural University,Guangzhou 510642
2. State Key Laboratory of Applied Microbiology in Southern China,Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,Guangdong Open Laboratory of Applied Microbiology,Guangdong Institute of Microbiology,Guangdong Academy of Sciences,Guangzhou 510070

Received:2020-11-14 Published:2021-08-26 Online:2021-09-10

摘要/Abstract

摘要：

利用染色体步移技术对植物内生真菌Paramyrothecium roridum中单端孢霉烯毒素的生物合成基因tri5、tri12启动子进行克隆,分别利用原核和真核表达系统以及荧光素酶表达系统对启动子核心区域进行鉴定,发现tri12的启动子片段12-f1对氨苄青霉素抗性基因和潮霉素抗性基因表达的启动效率最高,tri5的启动子片段5-f0对荧光素酶基因表达的启动效率最高;同时对启动子的功能组件进行分析,发现tri5含有TATA box和CAAT box,而tri12是TATA-less启动子,但含有CAAT box和GC box。本研究为强启动子的筛选及单端孢霉烯类化合物的高效生物合成奠定基础。

关键词: Paramyrothecium roridum, 单端孢霉烯族毒素, 启动子, 克隆, 鉴定

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

岑由飞, 朱牧孜, 叶伟, 李赛妮, 钟国华, 章卫民. Paramyrothecium roridum中单端孢霉烯毒素生物合成基因启动子的克隆和功能鉴定[J]. 生物技术通报, 2021, 37(8): 85-94.

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.

图/表 9

表1 目的基因tri5和tri12特异性反向引物序列

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

图1 tri5、tri12染色体步移扩增得到的目的片段 M：Marker trans2K plus;A：tri5染色体步移三轮PCR扩增产物;B：tri12染色体步移三轮PCR扩增产物

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

表2 目的基因tri5、tri12启动子核心序列

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

图2 构建具有不同长度片段的tri5和tri12基因启动子

图3 DH5α菌落PCR验证及启动子载体 M：Marker trans2K plus;A：DH5α-pET30a-5-f0-Amp菌落PCR图;B：pET30a-5-f0-Amp载体图谱;C：DH5α-YEp352-12-f2-HYRB-CYC1菌落PCR图;D：YEp352-12-f2-HYRB-CYC1载体图谱

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

图4 不同浓度的氨苄青霉素抗性平板筛选含不同启动子片段的大肠杆菌 A：DH5α-pET30a-5-f0/5-f1/5-f2/12-f0/12-f1/12-f2-Amp点板结果;B：DH5α-pET30a-5-f0/5-f1/5-f2-Amp点板结果;C：DH5α-YEp352-12-f1-HYRB-CYC1和DH5α-pET30a-12-f1-Amp的OD值柱形图。N：DH5α-pET30a-ACP空载（阴性对照）;P：DH5α-YEp352-12-f1-HYRB-CYC1（阳性对照）;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.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

图5 不同浓度的潮霉素抗性平板筛选含不同启动子片段的酿酒酵母 A：BJ5464-YEp352-TEF1-HYRB-CYC1（阳性对照）、BJ5464-YEp352-No pro-HYRB-CYC1（阴性对照）、BJ5464-YEp352-12-f1-HYRB-CYC1和BJ5464-YEp352-5-f0-HYRB-CYC1的点板结果;B：BJ5464-YEp352-TEF1-HYRB-CYC1（阳性对照）和BJ5464-YEp352-12-f1-HYRB-CYC1的OD值柱形图。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.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

图6 pGL3-5-f0-basic、pGL3-12-f1-basic和pGL3-pgpd-basic载体在DH5α中的荧光素酶活性检测

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

图7 tri5和tri12功能组件预测

Fig.7 Functional component prediction of gene tri5 and tri12

参考文献 20

[1]	Brown DW, Villani A, Susca A, et al. Gain and loss of a transcription factor that regulates late trichothecene biosynthetic pathway genes in Fusarium[J]. Fungal Genetics and Biology, 2020, 136:553-560.
[2]	Cardoza RE, McCormick SP, Lindo L, et al. A cytochrome P450 monooxygenase gene required for biosynjournal of the trichothecene toxin harzianum A in Trichoderma[J]. Applied Microbiology and Biotechnology, 2019, 103(19):8087-8103. doi: 10.1007/s00253-019-10047-2 pmid: 31384992
[3]	Lee SR, Seok S, Ryoo R, et al. Macrocyclic trichothecene mycotoxins from a deadly poisonous mushroom, Podostroma cornu-damae[J]. Journal of Natural Products, 2019, 82(1):122-128. doi: 10.1021/acs.jnatprod.8b00823 URL
[4]	Nguyen LTT, Jang JY, Kim TY, et al. Nematicidal activity of verrucarin A and roridin A isolated from Myrothecium verrucaria against Meloidogyne incognita[J]. Pesticide Biochemistry and Physiology, 2018, 148:133-143. doi: S0048-3575(17)30508-4 pmid: 29891364
[5]	Liu HX, Liu WZ, Chen YC, et al. Cytotoxic trichothecene macrolides from the endophyte fungus Myrothecium roridum[J]. Journal of Asian Natural Products Research, 2016, 18(7):684-692. doi: 10.1080/10286020.2015.1134505 URL
[6]	Ye W, Chen YC, Li HH, et al. Two trichothecene mycotoxins from Myrothecium roridum induce apoptosis of HepG-2 cells via caspase activation and disruption of mitochondrial membrane potential[J]. Molecules, 2016, 21(6):E781.
[7]	Cane DE. Enzymic formation of sesquiterpenes[J]. Chemical Reviews, 1990, 90(7):1089-1103. doi: 10.1021/cr00105a002 URL
[8]	McCormick SP, Alexander NJ, Trapp SE, et al. Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides[J]. Applied and Environmental Microbiology, 1999, 65(12):5252-5256. pmid: 10583973
[9]	Proctor RH, McCormick SP, Kim HS, et al. Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi[J]. PLoS Pathogens, 2018, 14(4):e1006946. doi: 10.1371/journal.ppat.1006946 URL
[10]	Trassaert M, Vandermies M, Carly F, et al. New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica[J]. Microbial Cell Factories, 2017, 16(1):141. doi: 10.1186/s12934-017-0755-0 pmid: 28810867
[11]	Cooper SJ, Trinklein ND, Anton ED, et al. Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome[J]. Genome Research, 2006, 16(1):1-10. pmid: 16344566
[12]	Even DY, Kedmi A, Ideses D, et al. Functional screening of core promoter activity[J]. Methods in Molecular Biology, 2017, 1651:77-91. doi: 10.1007/978-1-4939-7223-4_7 pmid: 28801901
[13]	Balabanova LA, Shkryl YN, Slepchenko LV, et al. Development of host strains and vector system for an efficient genetic transformation of filamentous fungi[J]. Plasmid, 2019, 101:1-9. doi: S0147-619X(18)30089-1 pmid: 30465791
[14]	朱利, 王义, 殷金瑶, 等. 橡胶树白粉菌内源强启动子WY193的克隆及功能鉴定[J]. 分子植物育种, 2020, 18(9):2865-2871.
	Zhu L, Wang Y, Yin JY, et al. Cloning and function identification of endogenous strong promoter WY193 of Oidium heveae steinm[J]. Molecular Plant Breeding, 2020, 18(9):2865-2871.
[15]	阎隆飞, 张玉麟. 分子生物学[M]. 第2 版. 北京: 中国农业大学出版社, 1997.
	Yan LF, Zhang YL. Molecular biology[M]. 2rd ed. Beijing: China Agricultural University Press, 1997.
[16]	Ye W, Liu TM, Zhu MZ, et al. De Novo transcriptome analysis of plant pathogenic fungus Myrothecium roridum and identification of genes associated with trichothecene mycotoxin biosynjournal[J]. International Journal of Molecular Sciences, 2017, 18(3):E497.
[17]	Ashrafi M, Fardsi M, Mirshamsi A, et al. Isolation and sequence analysis of gpdII promoter of the white button mushroom(Agaricus bisporus)from strains Holland737 and IM008[J]. International Journal of Horticultural Science and Technology, 2015, 2(1):33-41.
[18]	Madhavan A, Pandey A, et al. Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis[J]. Bioresource Technology, 2017, 245:1334-1342. doi: S0960-8524(17)30808-8 pmid: 28578805
[19]	Keränen S, Penttilä M. Production of recombinant proteins in the filamentous fungus Trichoderma reesei[J]. Current Opinion in Biotechnology, 1995, 6(5):534-537. pmid: 7579664
[20]	黄自磊, 章卫民, 叶伟, 等. 深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定[J]. 生物技术通报, 2018, 34(4):144-150.
	Huang ZL, Zhang WM, Ye W, et al. Cloning and identification of gene promoter for gliotoxin biosynjournal from deep-sea-derived fungus Dichotomomyces cejpii[J]. Biotechnology Bulletin, 2018, 34(4):144-150.

Paramyrothecium roridum中单端孢霉烯毒素生物合成基因启动子的克隆和功能鉴定

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

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