Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (6): 316-324.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1255
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DONG Cong(), GAO Qing-hua, WANG Yue, LUO Tong-yang, WANG Qing-qing
Received:
2022-10-11
Online:
2023-06-26
Published:
2023-07-07
Contact:
DONG Cong
E-mail:dongcong24@126.com
DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang, WANG Qing-qing. Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy[J]. Biotechnology Bulletin, 2023, 39(6): 316-324.
引物名称 Primer name | 基因 Gene | 引物序列 Primer sequence(5'-3') |
---|---|---|
H- F | HAC1 | CTCGCAGATCCTTGACTGAGGATCTGGACGAAG |
H- R | CCTCAGTCAAGGATCTGCGAGTGGATGTAGATG | |
E- F | Ero1 | TGGAGAAACAAATCTTTTACCGATTGGTTTCTG |
E- R | GGTAAAAGATTTGTTTCTCCAAACAGAGGTCTTC | |
P1-F | PDI | CTGCTGCCGAAATCTTAAAGGACAATGAGCAGG |
P1-R | CCTTTAAGATTTCGGCAGCAGAAACAAGTTCAG | |
P2-F | TTGACGGATCTACCTTCAAATCATATGCTGAAG | |
P2-F | ATTTGAAGGTAGATCCGTCAATGTCTCCAAATAG |
Table 1 Mutation primer sequences
引物名称 Primer name | 基因 Gene | 引物序列 Primer sequence(5'-3') |
---|---|---|
H- F | HAC1 | CTCGCAGATCCTTGACTGAGGATCTGGACGAAG |
H- R | CCTCAGTCAAGGATCTGCGAGTGGATGTAGATG | |
E- F | Ero1 | TGGAGAAACAAATCTTTTACCGATTGGTTTCTG |
E- R | GGTAAAAGATTTGTTTCTCCAAACAGAGGTCTTC | |
P1-F | PDI | CTGCTGCCGAAATCTTAAAGGACAATGAGCAGG |
P1-R | CCTTTAAGATTTCGGCAGCAGAAACAAGTTCAG | |
P2-F | TTGACGGATCTACCTTCAAATCATATGCTGAAG | |
P2-F | ATTTGAAGGTAGATCCGTCAATGTCTCCAAATAG |
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
RT GAP up | CCAGCGGCAACAAGATCAAC |
RT GAP down | CTCCTCGTTGACACCGACAA |
RT GDH up | GGCAATACCAATCTGTCAACCAACCTTA |
RT GDH down | AACCAGTGTTACCGATAGTTTCCCAAGC |
RT HAC1 up | CTGAATATGACGACGAAGAA |
RT HAC1 down | CTCCTGCTTGATAGATGTG |
RT PDI up | ACCACATTTTACGGAGTTGCCGGT |
RT PDI down | CCTCGCCAGGTCTGACAAGCA |
RT Ero1 up | ATGAGGATAGTAAGGAGCGTAGC |
RT Ero1 down | GTTGACCAGTTCCACCAGTTTG |
Table 2 Primers used in RT-qPCR
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
RT GAP up | CCAGCGGCAACAAGATCAAC |
RT GAP down | CTCCTCGTTGACACCGACAA |
RT GDH up | GGCAATACCAATCTGTCAACCAACCTTA |
RT GDH down | AACCAGTGTTACCGATAGTTTCCCAAGC |
RT HAC1 up | CTGAATATGACGACGAAGAA |
RT HAC1 down | CTCCTGCTTGATAGATGTG |
RT PDI up | ACCACATTTTACGGAGTTGCCGGT |
RT PDI down | CCTCGCCAGGTCTGACAAGCA |
RT Ero1 up | ATGAGGATAGTAAGGAGCGTAGC |
RT Ero1 down | GTTGACCAGTTCCACCAGTTTG |
菌株 Strain | GDH基因拷贝数 Copy number of GDH | HAC1基因拷贝数 Copy number of HAC1 | PDI基因拷贝数 Copy number of PDI | Ero1基因拷贝数 Copy number of Ero1 |
---|---|---|---|---|
CK(X33/GDH) | 3.20±0.20 | Endogenous 1 | Endogenous 1 | Endogenous 1 |
CK-H | 3.20±0.20 | 1.10±0.09 | Endogenous 1 | Endogenous 1 |
CK-2H | 3.20±0.20 | 2.08±0.26 | Endogenous 1 | Endogenous 1 |
CK-3H | 3.20±0.20 | 2.95±0.11 | Endogenous 1 | Endogenous 1 |
CK-4H | 3.20±0.20 | 4.35±0.21 | Endogenous 1 | Endogenous 1 |
CK-P | 3.20±0.20 | Endogenous 1 | 1.02±0.11 | Endogenous 1 |
CK-2P | 3.20±0.20 | Endogenous 1 | 2.32±0.19 | Endogenous 1 |
CK-3P(X33/GDH-3PDI) | 3.20±0.20 | Endogenous 1 | 3.006±0.20 | Endogenous 1 |
CK-4P | 3.20±0.20 | Endogenous 1 | 4.42±0.29 | Endogenous 1 |
CK-E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 1.21±0.13 |
CK-2E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 1.98±0.34 |
CK-3E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 3.22±0.17 |
CK-4E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 4.19±0.20 |
Table 3 Copy numbers of genes detected by RT-qPCR
菌株 Strain | GDH基因拷贝数 Copy number of GDH | HAC1基因拷贝数 Copy number of HAC1 | PDI基因拷贝数 Copy number of PDI | Ero1基因拷贝数 Copy number of Ero1 |
---|---|---|---|---|
CK(X33/GDH) | 3.20±0.20 | Endogenous 1 | Endogenous 1 | Endogenous 1 |
CK-H | 3.20±0.20 | 1.10±0.09 | Endogenous 1 | Endogenous 1 |
CK-2H | 3.20±0.20 | 2.08±0.26 | Endogenous 1 | Endogenous 1 |
CK-3H | 3.20±0.20 | 2.95±0.11 | Endogenous 1 | Endogenous 1 |
CK-4H | 3.20±0.20 | 4.35±0.21 | Endogenous 1 | Endogenous 1 |
CK-P | 3.20±0.20 | Endogenous 1 | 1.02±0.11 | Endogenous 1 |
CK-2P | 3.20±0.20 | Endogenous 1 | 2.32±0.19 | Endogenous 1 |
CK-3P(X33/GDH-3PDI) | 3.20±0.20 | Endogenous 1 | 3.006±0.20 | Endogenous 1 |
CK-4P | 3.20±0.20 | Endogenous 1 | 4.42±0.29 | Endogenous 1 |
CK-E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 1.21±0.13 |
CK-2E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 1.98±0.34 |
CK-3E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 3.22±0.17 |
CK-4E | 3.20±0.20 | Endogenous 1 | Endogenous 1 | 4.19±0.20 |
Fig. 2 Agarose gel electrophoresis analysis for pPICZB-HAC1n(n=1,2,3或4)(A),pPICZB-Ero1n(n=1,2,3或4)(B),pPICZB-PDIn(n=1,2,3或4)(C)digested with double enzymes M: DNA ladder; lanes 1-4: pPICZB-HAC1/Ero1/PDIn(n=1,2,3或4)digested with BamH I and Bgl II
Fig. 3 Relative enzyme activities of recombinant strains co-expressed with different molecular chaperones at test tube level The error line in the figure refers to the standard deviation. The lowercase letters indicate that the difference among different strains reached a significant level(P<0.05). The same below
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