生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 85-94.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1386
岑由飞1,2(), 朱牧孜2, 叶伟2, 李赛妮2, 钟国华1(), 章卫民2()
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: 基金资助:
CEN You-fei1,2(), ZHU Mu-zi2, YE Wei2, LI Sai-ni2, ZHONG Guo-hua1(), ZHANG Wei-min2()
Received:
2020-11-14
Published:
2021-08-26
Online:
2021-09-10
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。
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.
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 |
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
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 |
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
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