Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (8): 198-205.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1482
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TANG Guang-fu(), GUI Yan-ling, MAN Hai-qiao, ZHAO Jie-hong()
Received:
2021-11-29
Online:
2022-08-26
Published:
2022-09-14
Contact:
ZHAO Jie-hong
E-mail:1838921871@qq.com;105798711@qq.com
TANG Guang-fu, GUI Yan-ling, MAN Hai-qiao, ZHAO Jie-hong. Editing pyrG Gene of Monascus by CRISPR/Cas 9 and Its Effects on Secondary Metabolism[J]. Biotechnology Bulletin, 2022, 38(8): 198-205.
引物名称 Primer name | 引物或sgRNA序列 Primer or sgRNA sequence(5'-3') | 退火温度 Annea-ling temperature/℃ | 靶标序列长度 Target sequence length/bp |
---|---|---|---|
Cas9-F | GTTTAAGGTCCTGGGCAACA | 55 | 312 |
Cas9-R | ATCGTGGGGTACTTCTCGTG | ||
PyrGgr-F | GTTGAAACGAACCCCAGCAC | 57 | 380 |
pyrGgr-R | CCACATCGACATCCTCTCCG | ||
sgRNA | TTAATACGACTCACTATAGGGggcttgaagttcctgcgttgGTTTTAGAGCTAGAAATA | / | / |
Table 1 Primers and sgRNA sequences for PCR detection
引物名称 Primer name | 引物或sgRNA序列 Primer or sgRNA sequence(5'-3') | 退火温度 Annea-ling temperature/℃ | 靶标序列长度 Target sequence length/bp |
---|---|---|---|
Cas9-F | GTTTAAGGTCCTGGGCAACA | 55 | 312 |
Cas9-R | ATCGTGGGGTACTTCTCGTG | ||
PyrGgr-F | GTTGAAACGAACCCCAGCAC | 57 | 380 |
pyrGgr-R | CCACATCGACATCCTCTCCG | ||
sgRNA | TTAATACGACTCACTATAGGGggcttgaagttcctgcgttgGTTTTAGAGCTAGAAATA | / | / |
Fig. 3 Transformation of Cas 9 into monascus mediated by A. tumefaciens A:PCR detection of A. tumefaciens. B:Co-culture of A. tumefaciens and monascus. C:PCR detection of the Cas 9 of monascus chassis strain. 1-8:Different strains. M:DL1000 DNA marker
Fig. 4 Transformation of monascus protoplasts by sgRNA in vitro A:Inhibition of 5-FOA on monascus. B:Electrophoresis of the sgRNA. C:Prepared protoplasts. D:Screening resistant monascus
Fig. 5 Variation analysis of pyrG gene of monascus mutant A:Part nested peak sequence. B:PCR detection of pyrG gene in some strains. C:PCR product cutted by T7EI. D:Mutation loci of pyrG gene in some strains. MU:Mutant. WT:Wild-type. “+”:With DNA. “-”:Without DNA. Black box:Refers to the PAM site. Grey background:Refers to the sgRNA target sequence
Fig. 7 Effect of uracil riboside on the chemical composition content of different strains in medium A:Lovastatin absorbance. B:Monascus pigment content. C:Citrinin content. “-”:Not added. “+”:Added. WT:Wild type strain. MU:Mutant strain
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