鸡腿菇（Coprinus comatus）漆酶的诱导及cDNA克隆
摘要
漆酶是一种铜多酚氧化酶,主要存在于白腐菌中,是一种分泌至胞外的糖蛋白。漆酶可以降解木质素和氧化酚类、芳胺类化合物,有着广泛的用途。鸡腿菇是一种能高效分泌漆酶的白腐菌。
本论文首先研究了鸡腿菇在25℃、四种不同培养基中进行液体静置培养时,漆酶活力的比较。研究表明A培养基(碳源为葡萄糖,氮源为酵母浸膏)的鸡腿菇漆酶活性最高。后续实验研究了不同的金属离子和芳香族化合物对漆酶产酶的影响。结果显示添加浓度为0.8mmol/L的锰离子和3.0mmol/L的咖啡酸对鸡腿菇漆酶的诱导作用最明显。其它的化学试剂分别在不同的浓度表现出诱导作用。其中在低浓度表现出最佳诱导作用的是4-甲氧基肉桂酸、茴香酸、3,4,5-三甲氧基肉桂酸、愈创木酚、3,5-二甲氧基-4-羟基肉桂酸、香兰素、3-(4-甲氧苯基)-丙酸。而Cu~(2+)、3,4-二甲氧基苯甲醛、肉桂酸、4-羟基肉桂酸、3-(4-羟基苯基)-丙酸则在高浓度时诱导效果最好。对碳源和氮源的研究表明最佳碳源是葡萄糖,最佳氮源是蛋白胨。通过不连续聚丙酰烯酰胺凝胶电泳分析发现,不同种类、浓度的化学试剂会诱导产生不同的鸡腿菇漆酶的同工酶,由于这些同工酶具有不同的等电点所以电泳中表现出不同的谱带。漆酶最适反应pH值和温度为6.0和60℃。
本论文实验还从鸡腿菇中提取mRNA,反转录得到cDNA序列,再根据真菌漆酶蛋白质家族的保守氨基酸结构域设计一系列简并性引物,进行PCR扩增得到143bp的鸡腿菇漆酶特异性基因片段。再利用BLAST程序将该序列与NCBI数据库中的漆酶基因进行比对,证明与其它漆酶基因高度同源。再通过RACE(cDNA末端快速扩增技术)得到鸡腿菇漆酶5’端的cDNA序列,共626bp。
Laccase is a kind of copper–contained polyphenol oxidase produced mainly by white rot fungi and excreted into media in form of glycoprotein. It can degrade lignin and catalyze the oxidation of a broad number of phenolic compounds and aromatic amines. Laccase is applied in many areas. Coprinus comatus,a sort of white rot fungi produces laccase efficiently.
In this dissertation, laccase production of Coprinus comatus in four different media was studied under liquid culture at the temperature of 25℃. It was shown that the best medium was medium A which contained the glucose and yeast extract as carbon and nitrogen source respectively. The relation between the laccase production of Coprinus comatus and different metal ions and aromatic compounds was also discussed. It was shown the production of the laccase can be mostly induced by the addition of Mn2+ at concentration of 0.8 mmol/L and caffeic acid at the concentration of 3.0mmol/L. Other chemical agents contributed to the increase of the enzyme production most at different concentration. 4-methoxycinnamic acid, anisic acid, 3,4,5-trimethoxy-cinnamic acid, guaiacol, 3,5-dimethoxy-4-hydoxycinnamic acid, vanillin and 3-(4-methoxyphenyl)propionic acid induced the enzyme production most at low concentration, however, Cu2+, 3,4-dimethoxybenzaldehyde, cinnamic acid,4-hydroxycinnamic acid, and 3-(4-hydroxyphenyl)propionic acid did at high concentration. The result also showed that glucose was the best carbon source and peptone was the best nitrogen source. From the discontinuous polyacrylamide gel electrophoresis, we found different compounds at diverse concentration induced many laccase isoenzymes of Coprinus comatus displayed different bands with various pI. The optimum pH value and temperature of reaction of laccase was 6.0 and 60℃.
In this paper, extraction of total RNA from Coprinus comatus and RT technology was used to gain the cDNA sequences. A series of degenerate oligonucleotide primers were designed , coding for conservative amino acid regions in fungi laccase protein family. A PCR amplification was performed on Coprinus comatus cDNA template and a specific fragment of 143 bp was obtained. By using the program of BLAST on NCBI GenBank database, the sequence presented a high match with the laccase gene from other fungi. The cDNA sequences of 5′ends were isolated by using RACE(rapid amplification of cDNA ends) technique from Coprinus comatus.The length of sequence was 626bp.
本论文首先研究了鸡腿菇在25℃、四种不同培养基中进行液体静置培养时,漆酶活力的比较。研究表明A培养基(碳源为葡萄糖,氮源为酵母浸膏)的鸡腿菇漆酶活性最高。后续实验研究了不同的金属离子和芳香族化合物对漆酶产酶的影响。结果显示添加浓度为0.8mmol/L的锰离子和3.0mmol/L的咖啡酸对鸡腿菇漆酶的诱导作用最明显。其它的化学试剂分别在不同的浓度表现出诱导作用。其中在低浓度表现出最佳诱导作用的是4-甲氧基肉桂酸、茴香酸、3,4,5-三甲氧基肉桂酸、愈创木酚、3,5-二甲氧基-4-羟基肉桂酸、香兰素、3-(4-甲氧苯基)-丙酸。而Cu~(2+)、3,4-二甲氧基苯甲醛、肉桂酸、4-羟基肉桂酸、3-(4-羟基苯基)-丙酸则在高浓度时诱导效果最好。对碳源和氮源的研究表明最佳碳源是葡萄糖,最佳氮源是蛋白胨。通过不连续聚丙酰烯酰胺凝胶电泳分析发现,不同种类、浓度的化学试剂会诱导产生不同的鸡腿菇漆酶的同工酶,由于这些同工酶具有不同的等电点所以电泳中表现出不同的谱带。漆酶最适反应pH值和温度为6.0和60℃。
本论文实验还从鸡腿菇中提取mRNA,反转录得到cDNA序列,再根据真菌漆酶蛋白质家族的保守氨基酸结构域设计一系列简并性引物,进行PCR扩增得到143bp的鸡腿菇漆酶特异性基因片段。再利用BLAST程序将该序列与NCBI数据库中的漆酶基因进行比对,证明与其它漆酶基因高度同源。再通过RACE(cDNA末端快速扩增技术)得到鸡腿菇漆酶5’端的cDNA序列,共626bp。
Laccase is a kind of copper–contained polyphenol oxidase produced mainly by white rot fungi and excreted into media in form of glycoprotein. It can degrade lignin and catalyze the oxidation of a broad number of phenolic compounds and aromatic amines. Laccase is applied in many areas. Coprinus comatus,a sort of white rot fungi produces laccase efficiently.
In this dissertation, laccase production of Coprinus comatus in four different media was studied under liquid culture at the temperature of 25℃. It was shown that the best medium was medium A which contained the glucose and yeast extract as carbon and nitrogen source respectively. The relation between the laccase production of Coprinus comatus and different metal ions and aromatic compounds was also discussed. It was shown the production of the laccase can be mostly induced by the addition of Mn2+ at concentration of 0.8 mmol/L and caffeic acid at the concentration of 3.0mmol/L. Other chemical agents contributed to the increase of the enzyme production most at different concentration. 4-methoxycinnamic acid, anisic acid, 3,4,5-trimethoxy-cinnamic acid, guaiacol, 3,5-dimethoxy-4-hydoxycinnamic acid, vanillin and 3-(4-methoxyphenyl)propionic acid induced the enzyme production most at low concentration, however, Cu2+, 3,4-dimethoxybenzaldehyde, cinnamic acid,4-hydroxycinnamic acid, and 3-(4-hydroxyphenyl)propionic acid did at high concentration. The result also showed that glucose was the best carbon source and peptone was the best nitrogen source. From the discontinuous polyacrylamide gel electrophoresis, we found different compounds at diverse concentration induced many laccase isoenzymes of Coprinus comatus displayed different bands with various pI. The optimum pH value and temperature of reaction of laccase was 6.0 and 60℃.
In this paper, extraction of total RNA from Coprinus comatus and RT technology was used to gain the cDNA sequences. A series of degenerate oligonucleotide primers were designed , coding for conservative amino acid regions in fungi laccase protein family. A PCR amplification was performed on Coprinus comatus cDNA template and a specific fragment of 143 bp was obtained. By using the program of BLAST on NCBI GenBank database, the sequence presented a high match with the laccase gene from other fungi. The cDNA sequences of 5′ends were isolated by using RACE(rapid amplification of cDNA ends) technique from Coprinus comatus.The length of sequence was 626bp.
引文
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