平菇遗传转化体系和转漆酶工程菌株的构建
摘要
我国每年仅农业生产中形成的农作物残渣(麦秸、玉米秸秆)等就约6亿t,其富含木质纤维素。应用食用菌转化秸秆、变废为宝是一条成功的途径。为提高食用菌利用木质纤维素的能力,提高食用菌对秸秆的生物学效率,本文以平菇天达300为材料,构建了高效遗传表达体系,并进行了漆酶基因转化。研究结果如下:
(1)平菇sdi启动子的克隆及表达载体25号的构建和转化
克隆了平菇天达300的同源启动子-1.3kb的sdi启动子,利用重叠PCR将sdi启动子片段和潮霉素抗性基因hph融合,构建了以pMD19-T载体为骨架,含有sdi启动子和hph以及CaMV终止子的表达载体25号。
以液体培养的平菇菌丝为实验材料,0.6mol/L甘露醇作渗透压稳定剂,浓度为1.6%的溶壁酶溶液,30℃酶解3h,获得具有再生活力的原生质体。平菇天达300原生质体的得率约为5.7×10~6个/mL,在RCM再生平板上的再生率为0.46%。
通过平菇的潮霉素抗性试验,确定了其菌丝和原生质体的潮霉素致死浓度。据此将转化的初筛浓度定为80μg/mL,复筛浓度定为100μg/mL。采用PEG-CaCl_2介导原生质体转化的方法,将表达载体25号转化入平菇,在含80μg/mL潮霉素RCM平板上,获得一系列转化子。这些转化子经过复筛和PCR鉴定后得到潮霉素抗性基因hph整合入宿主基因组的阳性转化子。将阳性转化子菌株进行表型和RNA转录水平验证,表明hph基因整合入平菇的基因组,其抗性可以稳定的遗传和表达,从而建立了平菇的遗传转化体系。
(2)食用菌不同转化体系转化效率的比较
将两个含有潮霉素抗性基因但启动子不同的真菌表达质粒pAN7-1和PBHt1,导入平菇中与25号做比较表明,不同的启动子得到的抗性转化子生长速率、转化率、RNA水平上的转录都不同。pAN7-1抗性转化子转化率最高,0.4个转化子/μg质粒DNA,其hph目的基因的表达量最大;其次是25号抗性质粒,0.2个转化子/μg质粒DNA;PBHt1的表达量较少。
(3)平菇漆酶基因poxc的克隆和同源表达
利用RT-PCR和LA PCR步行等技术从平菇天达300中获得编码漆酶基因poxc的gDNA和cDNA。gDNA全长为3818bp,包含19个内含子和20个外显子。cDNA序列全长为1607bp,编码533个氨基酸的蛋白,与其它真菌漆酶蛋白序列有较高的同源性,并且含有3个真菌类Cu-oxidase的高度保守结构域。
构建了含有sdi启动子和poxc基因的同源表达载体PIPOXC和PI2POXC,和25号抗性表达载体共转化平菇的原生质体,获得了具有潮霉素抗性的假定转化子21个,但是经PCR检测后发现poxc基因未完全整合入平菇基因组,漆酶酶活提高不明显,没有筛选到预期的漆酶活性较高的转化子。
There were about 600 million tons crop residues produced per year in China. They were rich in lignocelluloses. It was a successful approach to turn crop residues into wealth food using edible fungi. In present study, we constructed an effective genetic transformation system for transformation laccase gene into Pleurotus ostreatus Tianda 300 to enhance its capacity of decomposing the lignocelluloses from crop residues and the biological efficiency. The results were as follows:
(1) Cloning of sdi promoter of Pleurotus ostreatus and construction of expression Vector 25
Homologous sdi promoters with 1.3 kb of Pleurotus ostreatus tianda 300 was cloned and fused with gene hph by overlapping PCR. The hygromycin resistance expression vector 25 was constructed, which was constituted of sdi promoter, hph gene and CaMV termination in skeleton of pMD19 - T carrie.
Protoplasts with regeneration ability of Pleurotus ostreatus tianda 300 were obtained by hydrolysis mycelium using 1.6% lyticase enzyme in 30℃for 3h, and 0.6 mol/L mannitol as osmotic pressure stabilizer. The protoplast yield was about 5.7 X 106/ mL, and regeneration rate on RCM plate was 0.46%.
The hygromycin resistance concentration of mycelium and protoplast of Pleurotus ostreatus tianda 300 was determined, and base on it hygromycin concentration of preliminary and counter screening for transformation was set up as 80μg/mL and 100μg/mL, respectively. The expression vector 25 was transformed into mushroom by PEG-CaCl_2 mediated protoplast method, and some transformants grew on RCM plate with 80μg/mL hygromycin. These transformants were all identified as positive ones through the screening test, gene DNA PCR, and mRNA RT-PCR. It was proved that the hph gene integrated into the genome and expressed stability. Thereby, we established a genetic transformation system of Pleurotus ostreatus.
(2) Compare between different transformation systems of Pleurotus ostreatus
The transformations frequency of three expression vectors 25 and pAN7-1 into Pleurotus ostreatus tianda 300 were 0.2 transformants/μg plasmid DNA, 0.4 transformants/μg plasmid DNA, respectively, and the lowest of PBHt1. The RNA transcription levels of hph gene of these three expression vectors were similar with their transformations frequency.
(3) Cloning and homologous expression of laccase poxc gene of Pleurotus ostreatus
The gDNA and cDNA of laccase poxc gene of Pleurotus ostreatus tianda 300 were cloned. The gDNA length was 3818bp, including 19 introns and 20 exons. The cDNA length was 1607bp, encoding 533 amino acid protein, containing three highly conserved Cu-oxidase domains homologized higher with other fungal laccase protein.
Laccase poxc homologous expression vector PIPOXC and PI2POXC of Pleurotus ostreatus were constructed by using gDNA and cDNA of Pleurotus ostreatus tianda 300 replace hph gene of expression vectors 25, and co-transformation with expression vectors 25 into Pleurotus ostreatus protoplast by PEG-CaCl_2 mediated, respectively. 21 hygromycin transformants were obtained. But we found poxc did not fully integrate into the genome by PCR detection, laccase activities did not differ from start strain. There were not any positive transformants obtained.
(1)平菇sdi启动子的克隆及表达载体25号的构建和转化
克隆了平菇天达300的同源启动子-1.3kb的sdi启动子,利用重叠PCR将sdi启动子片段和潮霉素抗性基因hph融合,构建了以pMD19-T载体为骨架,含有sdi启动子和hph以及CaMV终止子的表达载体25号。
以液体培养的平菇菌丝为实验材料,0.6mol/L甘露醇作渗透压稳定剂,浓度为1.6%的溶壁酶溶液,30℃酶解3h,获得具有再生活力的原生质体。平菇天达300原生质体的得率约为5.7×10~6个/mL,在RCM再生平板上的再生率为0.46%。
通过平菇的潮霉素抗性试验,确定了其菌丝和原生质体的潮霉素致死浓度。据此将转化的初筛浓度定为80μg/mL,复筛浓度定为100μg/mL。采用PEG-CaCl_2介导原生质体转化的方法,将表达载体25号转化入平菇,在含80μg/mL潮霉素RCM平板上,获得一系列转化子。这些转化子经过复筛和PCR鉴定后得到潮霉素抗性基因hph整合入宿主基因组的阳性转化子。将阳性转化子菌株进行表型和RNA转录水平验证,表明hph基因整合入平菇的基因组,其抗性可以稳定的遗传和表达,从而建立了平菇的遗传转化体系。
(2)食用菌不同转化体系转化效率的比较
将两个含有潮霉素抗性基因但启动子不同的真菌表达质粒pAN7-1和PBHt1,导入平菇中与25号做比较表明,不同的启动子得到的抗性转化子生长速率、转化率、RNA水平上的转录都不同。pAN7-1抗性转化子转化率最高,0.4个转化子/μg质粒DNA,其hph目的基因的表达量最大;其次是25号抗性质粒,0.2个转化子/μg质粒DNA;PBHt1的表达量较少。
(3)平菇漆酶基因poxc的克隆和同源表达
利用RT-PCR和LA PCR步行等技术从平菇天达300中获得编码漆酶基因poxc的gDNA和cDNA。gDNA全长为3818bp,包含19个内含子和20个外显子。cDNA序列全长为1607bp,编码533个氨基酸的蛋白,与其它真菌漆酶蛋白序列有较高的同源性,并且含有3个真菌类Cu-oxidase的高度保守结构域。
构建了含有sdi启动子和poxc基因的同源表达载体PIPOXC和PI2POXC,和25号抗性表达载体共转化平菇的原生质体,获得了具有潮霉素抗性的假定转化子21个,但是经PCR检测后发现poxc基因未完全整合入平菇基因组,漆酶酶活提高不明显,没有筛选到预期的漆酶活性较高的转化子。
There were about 600 million tons crop residues produced per year in China. They were rich in lignocelluloses. It was a successful approach to turn crop residues into wealth food using edible fungi. In present study, we constructed an effective genetic transformation system for transformation laccase gene into Pleurotus ostreatus Tianda 300 to enhance its capacity of decomposing the lignocelluloses from crop residues and the biological efficiency. The results were as follows:
(1) Cloning of sdi promoter of Pleurotus ostreatus and construction of expression Vector 25
Homologous sdi promoters with 1.3 kb of Pleurotus ostreatus tianda 300 was cloned and fused with gene hph by overlapping PCR. The hygromycin resistance expression vector 25 was constructed, which was constituted of sdi promoter, hph gene and CaMV termination in skeleton of pMD19 - T carrie.
Protoplasts with regeneration ability of Pleurotus ostreatus tianda 300 were obtained by hydrolysis mycelium using 1.6% lyticase enzyme in 30℃for 3h, and 0.6 mol/L mannitol as osmotic pressure stabilizer. The protoplast yield was about 5.7 X 106/ mL, and regeneration rate on RCM plate was 0.46%.
The hygromycin resistance concentration of mycelium and protoplast of Pleurotus ostreatus tianda 300 was determined, and base on it hygromycin concentration of preliminary and counter screening for transformation was set up as 80μg/mL and 100μg/mL, respectively. The expression vector 25 was transformed into mushroom by PEG-CaCl_2 mediated protoplast method, and some transformants grew on RCM plate with 80μg/mL hygromycin. These transformants were all identified as positive ones through the screening test, gene DNA PCR, and mRNA RT-PCR. It was proved that the hph gene integrated into the genome and expressed stability. Thereby, we established a genetic transformation system of Pleurotus ostreatus.
(2) Compare between different transformation systems of Pleurotus ostreatus
The transformations frequency of three expression vectors 25 and pAN7-1 into Pleurotus ostreatus tianda 300 were 0.2 transformants/μg plasmid DNA, 0.4 transformants/μg plasmid DNA, respectively, and the lowest of PBHt1. The RNA transcription levels of hph gene of these three expression vectors were similar with their transformations frequency.
(3) Cloning and homologous expression of laccase poxc gene of Pleurotus ostreatus
The gDNA and cDNA of laccase poxc gene of Pleurotus ostreatus tianda 300 were cloned. The gDNA length was 3818bp, including 19 introns and 20 exons. The cDNA length was 1607bp, encoding 533 amino acid protein, containing three highly conserved Cu-oxidase domains homologized higher with other fungal laccase protein.
Laccase poxc homologous expression vector PIPOXC and PI2POXC of Pleurotus ostreatus were constructed by using gDNA and cDNA of Pleurotus ostreatus tianda 300 replace hph gene of expression vectors 25, and co-transformation with expression vectors 25 into Pleurotus ostreatus protoplast by PEG-CaCl_2 mediated, respectively. 21 hygromycin transformants were obtained. But we found poxc did not fully integrate into the genome by PCR detection, laccase activities did not differ from start strain. There were not any positive transformants obtained.
引文
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