Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (8): 85-94.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1386
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CEN You-fei1,2(), ZHU Mu-zi2, YE Wei2, LI Sai-ni2, ZHONG Guo-hua1(), ZHANG Wei-min2()
Received:
2020-11-14
Online:
2021-08-26
Published:
2021-09-10
Contact:
ZHONG Guo-hua,ZHANG Wei-min
E-mail:297288514@qq.com;guohuazhong@scau.edu.cn;wmzhang@gdim.cn
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 |
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
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 |
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
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