柱状田头菇遗传转化体系启动子的筛选

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

摘要/Abstract

摘要：

柱状田头菇（茶树菇）Cyclocybe aegerita是一种美味的食用菌,具有很高的经济价值。构建高效表达的遗传转化体系,可以深入揭示其遗传特征及基因功能。以柱状田头菇YSG为实验材料,使用多片段重组克隆构建载体,PEG介导的原生质体转化,并以qRT-PCR为测定方法,对actin、gpd和Pumgpd不同长度5个启动子片段驱动靶标基因的表达量进行分析。依据actin基因启动子构建的载体pAa-actin-1和pAa-actin-2获得转化子中,靶标基因表达量为对照3倍以上转化子数量分别为50%和33.33%,最高表达量分别为对照的10.45和6.23倍;携带pAa-gpd-1、pAa-gpd-2和pAa-Pumgpd启动子的载体获得的转化子中,表达量为对照3倍以上的数量分别为0、28.57%和25%,最高表达量分别为对照的2.93、7.75和4.31倍。actin启动子获得高表达转化子得率较gpd和Pumgpd启动子片段高,适用于柱状田头菇转化体系构建。

关键词: 柱状田头菇, 启动子筛选, 遗传转化, 载体构建, 过量表达

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

崔祥华, 陶南, 程波普, 赵永昌, 陈卫民, 李靖. 柱状田头菇遗传转化体系启动子的筛选[J]. 生物技术通报, 2021, 37(5): 259-266.

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.

图/表 8

表1 载体构建和基因表达引物及其序列

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

图1 载体构建示意图重组质粒的LB、RB区间。LB和RB：农杆菌T-DNA的左边界和右边界

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

图2 启动子与mek片段扩增 M：DL2000 marker;A：1-2分别为启动子actin-1、gpd-1;B：1-3分别为actin-2、gpd-2和Pumgpd;C：1-5分别为Mek-A1、Mek-G1、Mek-A2、Mek-G2和Mek-PG片段

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

图3 启动子序列元件特征 A：actin启动子序列;B：gpd启动子序列;C：Pumgpd启动子序列。边框序列表示启动子元件

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

图4 重组质粒中转化片段扩增 M：DL10000 DNA marker; 1-5：为pAa-actin-1、pAa-actin-2、pAa-gpd-1、pAa-gpd-2和pAa-Pumgpd长片段

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

图5 潮霉素抗性平板上转化子的筛选 A：转化子的初选;a：对照;b-f：转化子;箭头指示长出的转化子。B：转化子的复筛;g：野生型YSG;h-l：复筛转化子

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

图6 部分转化子PCR验证 M：DL5000 DNA marker;1-4：pAa-actin-1转化子;5-8：pAa-actin-2转化子;9-12：pAa-gpd-1转化子;13-16：pAa-gpd-2转化子;17-20：pAa-Pumgpd转化子

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

图7 5个启动子片段控制的mek基因表达水平

Fig.7 Expression levels of mek gene controlled by five promoter fragments

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