Screening Promoters for Genetic Transformation of Cyclocybe aegerita

doi:10.13560/j.cnki.biotech.bull.1985.2020-1404

Abstract

Abstract:

Cyclocybe aegerita is a delicious fungus with high economic value. The genetic characterization and gene function of C. aegerita can be uncovered through the construction of highly expressed genetic transformation system. C. aegerita strain YSG was employed in this study,and the multi-fragments recombination and cloning were used to construct a plasmid,and protoplast was transformed by PEG mediation. The expression levels of targeting gene driving by 5 varied-length promoter fragments of actin,gpd and Pumgpd were analyzed via qRT-PCR. Among the transformants carrying plasmid pAa-actin-1 and pAa-actin-2 constructed by promoter elements of actin,the numbers of the transformants with the targeting gene expression level more than 3 times were 50.00% and 33.33% respectively,with the highest expressions to 10.45 and 6.23 times of the control,respectively. Accordingly,the numbers of the transformants carrying pAa-gpd-1,pAa-gpd-2 and pAa-Pumgpd with expression level more than 3 times were 0,28.57% and 25.00% with the highest expression levels to 2.93,7.75 and 4.31 times of the control,respectively. In conclusion,the yield of transformants with high expression level of targeting gene driven by actin promoter is higher than that by gpd and Pumgpd,indicating that actin promoter is suitable for the construction of genetic transformation system of C. aegerita.

Key words: Cyclocybe aegerita, promoter screening, genetic transformation, plasmid construction, overexpression

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.

Figures/Tables 8

References 20

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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

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