Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 259-266.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1404
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CUI Xiang-hua1,2(), TAO Nan2, CHENG Bo-pu1,2, ZHAO Yong-chang2, CHEN Wei-min2, LI Jing1()
Received:
2020-11-18
Online:
2021-05-26
Published:
2021-06-11
Contact:
LI Jing
E-mail:cxh.1994@qq.com;lijingcas@163.com
CUI Xiang-hua, TAO Nan, CHENG Bo-pu, ZHAO Yong-chang, CHEN Wei-min, LI Jing. Screening Promoters for Genetic Transformation of Cyclocybe aegerita[J]. Biotechnology Bulletin, 2021, 37(5): 259-266.
引物Primer | 序列Sequence(5'-3') | 用途 Function |
---|---|---|
actin-1-F | gagacggtccagcatctgcagCTCATACCCCGCTCTCACCG | actin-1扩增 |
actin-1-R | cgtttcaggaccatGGCTATTATTCGTTACGGTGAAGA | |
gpd-1-F | gagacggtccagcatctgcagGGCGCCCAGTAGTTGGGA | gpd-1扩增 |
gpd-1-R | ttcaggaccatCGCCGTAAACGAGAGATCTAGC | |
actin-2-F | gagacggtccagcatctgcagGTACAGGATCCTGAGTTTGTTAACCTT | actin-2扩增 |
actin-2-R | cgtttcaggaccatGGCTATTATTCGTTACGGTGAAGA | |
gpd-2-F | gagacggtccagcatctgcagCACTGGGTTGCTGGCCAA | gpd-2扩增 |
gpd-2-R | ttcaggaccatCGCCGTAAACGAGAGATCTAGC | |
Pumgpd-F | gagacggtccagcatctgcagAGATCTTGCTGATAGGCAGGTTTG | Pumgpd扩增 |
Pumgpd-R | gcgtttcaggaccatTATGGAAGAGTGTTTTGGTTTCGA | |
Mek-A1-F | atagccATGGTCCTGAAACGCAAACG | Mek-A1扩增 |
Mek-A1-R | tcgcccttcgagaccggatccTCAATTATCGTCATAAGCCTGGAA | |
Mek-G1-F | tttacggcgATGGTCCTGAAACGCAAACG | Mek-G1扩增 |
Mek-G1-R | tcgcccttcgagaccggatccTCAATTATCGTCATAAGCCTGGAA | |
Mek-A2-F | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | Mek-A2扩增 |
Mek-A2-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
Mek-G2-F | tttacggcgATGGTCCTGAAACGCAAACG | Mek-G2扩增 |
Mek-G2-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
Mek-PG-F | ccataATGGTCCTGAAACGCAAACG | Mek-PG扩增 |
Mek-PG-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
pAa-F | GAACGAGGCTTATGTCCACT | 转化片段扩增 |
pAa -R | GGTCTAGCCTGGTCACTGTC | |
PM-F | AAGAGGTGCTATGGGCTACG | 转化子验证 |
PM-R | CCCTTATCTGGGAACTACTCAC | |
qMek-F | GCCACACTGCCCACAGAATA | mek基因表达水平 |
qMek-R | AGGCTGACTTGAAGCACTTTC | |
qgpd-F | TCATCAATGGCAAGCCTG | 内参基因表达水平 |
qgpd-R | CCAAGTTCACACCACAGACG |
Table1 Primer sequences for plasmid construction and gene expression
引物Primer | 序列Sequence(5'-3') | 用途 Function |
---|---|---|
actin-1-F | gagacggtccagcatctgcagCTCATACCCCGCTCTCACCG | actin-1扩增 |
actin-1-R | cgtttcaggaccatGGCTATTATTCGTTACGGTGAAGA | |
gpd-1-F | gagacggtccagcatctgcagGGCGCCCAGTAGTTGGGA | gpd-1扩增 |
gpd-1-R | ttcaggaccatCGCCGTAAACGAGAGATCTAGC | |
actin-2-F | gagacggtccagcatctgcagGTACAGGATCCTGAGTTTGTTAACCTT | actin-2扩增 |
actin-2-R | cgtttcaggaccatGGCTATTATTCGTTACGGTGAAGA | |
gpd-2-F | gagacggtccagcatctgcagCACTGGGTTGCTGGCCAA | gpd-2扩增 |
gpd-2-R | ttcaggaccatCGCCGTAAACGAGAGATCTAGC | |
Pumgpd-F | gagacggtccagcatctgcagAGATCTTGCTGATAGGCAGGTTTG | Pumgpd扩增 |
Pumgpd-R | gcgtttcaggaccatTATGGAAGAGTGTTTTGGTTTCGA | |
Mek-A1-F | atagccATGGTCCTGAAACGCAAACG | Mek-A1扩增 |
Mek-A1-R | tcgcccttcgagaccggatccTCAATTATCGTCATAAGCCTGGAA | |
Mek-G1-F | tttacggcgATGGTCCTGAAACGCAAACG | Mek-G1扩增 |
Mek-G1-R | tcgcccttcgagaccggatccTCAATTATCGTCATAAGCCTGGAA | |
Mek-A2-F | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | Mek-A2扩增 |
Mek-A2-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
Mek-G2-F | tttacggcgATGGTCCTGAAACGCAAACG | Mek-G2扩增 |
Mek-G2-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
Mek-PG-F | ccataATGGTCCTGAAACGCAAACG | Mek-PG扩增 |
Mek-PG-R | tcgagaccggatccgccatggTCAATTATCGTCATAAGCCTGGAA | |
pAa-F | GAACGAGGCTTATGTCCACT | 转化片段扩增 |
pAa -R | GGTCTAGCCTGGTCACTGTC | |
PM-F | AAGAGGTGCTATGGGCTACG | 转化子验证 |
PM-R | CCCTTATCTGGGAACTACTCAC | |
qMek-F | GCCACACTGCCCACAGAATA | mek基因表达水平 |
qMek-R | AGGCTGACTTGAAGCACTTTC | |
qgpd-F | TCATCAATGGCAAGCCTG | 内参基因表达水平 |
qgpd-R | CCAAGTTCACACCACAGACG |
Fig. 1 Schematic diagram of plasmid construction LB and RB intervals of the recombinant plasmid. LB and RB:Left border and right border sequences of Agrobacterium T-DNA
Fig.2 Amplification fragments of promoters and mek M: DL2000 marker. A: 1-2: Promoter actin-1, gpd-1. B: 1-3:Promoter actin-2, gpd-2 and Pumgpd.C: 1-5:Fragment Mek-A1, Mek-G1, Mek-A2, Mek-G2 and Mek-PG
Fig.3 Characterization of promoter sequences and elements A: actin promoter sequence. B: gpd promoter sequence. C: Pumgpd promoter sequence. The promoter elements shown in the box
Fig.4 Amplification of transformation fragments in the recombinant plasmids M: DL10000 DNA marker. 1-5:The large fragments of pAa-actin-1, pAa-actin-2, pAa-gpd-1, pAa-gpd-2 and pAa-Pumgpd
Fig.5 Screening of transformants on HYG plates A: Primary screening of transformants, a: the control, b-f: transformants, the arrows show the transformants. B: Rescreening of transformants, g: wild type, h-l: Rescreening transformants
Fig.6 PCR verification of partial transformants M:DL5000 DNA marker. 1-4:transformants of pAa-actin-1. 5-8:transformants of pAa-actin-2. 9-12:transformants of pAa-gpd-1. 13-16:transformants of pAa-gpd-2. 17-20:transformants of pAa-Pumgpd
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