抗甲醛微生物的分离鉴定及其相关基因的克隆
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摘要
本研究从家具厂未处理污水中分离具有甲醛抗性的细菌和真菌。主要研究结果如下:
1.抗甲醛细菌的分离与鉴定
分离了8株抗甲醛细菌,6个菌株能在20mmol/L甲醛的平板生长,命名为SG-32、SG-71、SG-87、SG-107、SG-122、SG-135。1个能在10mmol/L的甲醛平板上生长,命名为SG-zong。1个能在4mmol/L甲醛平板上生长,命名为SG-ju。经生理生化分析和分子鉴定,初步确定SG-32、SG-71、SG-87、SG-107、SG-122和SG-135是Pseudomonas putida恶臭假单胞菌。SG-zong是Pseudomonas fluorescens荧光假单胞菌。SG-ju是Acinetobacter johnsonii.约氏不动杆菌。
2.抗甲醛细菌生长特性和抗甲醛能力分析
SG-32、SG-71和SG-107最适生长温度为30℃,SG-135则是35℃。SG-32和SG-135的最适pH值为pH7。SG-71和SG-107的最适pH值为pH8。这4株菌可以耐受并代谢20 -35mmol/l的甲醛。
3.抗甲醛真菌的分离与鉴定
分离了3种抗甲醛真菌,SG-huang、SG-hei和SG-qing。通过形态学观察和分子鉴定,初步确定它们分别是Aspergillus nomius红绶曲霉、Aspergillus versicolor花斑曲霉和Penicillium vinaceum酒色青霉。
4.抗甲醛真菌的生长特性和抗甲醛能力分析
SG-huang、SG-he和SG-qing最适生长温度均为25℃。最适pH值为pH5。这3种真菌都可以降解甲醛,其中SG-huang7天内可降解80mmol/L甲醛,SG-hei7天内可降解50mmol/L甲醛,SG-qing7天内可降解30mmol/L甲醛。它们还能以甲醛作为唯一碳元进行代谢。
5.抗甲醛细菌Gfa基因的克隆
利用PCR法克隆了Pseudomonas putida strain SG-32的Gfa基因。该基因核苷酸序列全长为393bp,编码131个氨基酸。在GenBank进行了登录,序列号为HM016790。BLAST检索分析表明,Pseudomonas putida strain SG-32 Gfa基因与与Pseudomonas putida F1中依赖谷胱甘肽的甲醛激活酶(glutathione-dependent formaldehyde-activating,Gfa)序列同源性96%。该蛋白有4个区域,一个VWFC区,1个C-半胱氨酸节区,1个微管蛋白亚基α、β和γ识别区,1个过敏毒素区。Pseudomonas putida strain SG-32 Gfa基因的克隆,为今后利用该基因进行抗甲醛基因工程奠定了基础。
In this study, formaldehyde-resistanced bacterias and fungis were isolated from furniture factory sewage as the experimental material.The results were as follows.
1,Isolated and identified the formaldehyde-resistanced bacterias
8 strains formaldehyde-resistanced bacterias were isolated on selective media contaning formaldehyde.There are 6 isolated strains named as SG-32,SG-71,SG-87,SG-107,SG-122,SG-135 could grow on medium with 20mmol/L formaldehyde.One isolated strain named as SG-zong could grow on medium with 10mmol/L formaldehyde,and the anther one strain named as SG-ju could grow on medium with 4mmol/L formaldehyde.SG-32,SG-71,SG-87,SG-107,SG-122,SG-135 were identified as Pseudomonas putida by plentiful physiological-biochemical experiments and molecular identification.SG-zong was identified as Pseudomonas fluorescen.SG-ju was identified as Acinetobacter johnsonii.
2,Analysis the growth and the ability of formaldehyde-resistanced of bacterias
The optimal temperature were 30℃for SG-32,SG-71,SG-107 respectively and which were 35℃for SG-135. The optimal pH value were 7 for SG-32,SG-135 respectively and which were 8 for SG-71,SG-107.They all could tolerated 20mmol/L-35mmol/L formaldehyde.
3,Isolated and identified the formaldehyde-resistanced fungis
Formaldehyde-degrading fungis(SG-huang,SG-hei,SG-qing) were isolated and identified as Aspergillus nomius,Aspergillus versicolor,Penicillium vinaceum by morphological observation and molecular identification.
4,Analysis the growth and the ability of formaldehyde-resistanced of fungis
The optimal temperature were 25℃for SG-huang,SG-he,SG-qing respectively.The optimal pH value were 5 for SG-huang,SG-he,SG-qing respectively.After shaking culturing 7 days, formaldehyde degraded from 80, 50, 30mmol/L to almost 0 mmol/L bySG-huang,SG-he,SG-qing respectively.All the isolated strains could grow on medium with formaldehyde as the sole carbon source.
5,Cloning of Gfa from the formaldehyde-resistanced bacteria
The DNA sequence of Gfa gene from Pseudomonas putida strain SG-32 was determined by PCR.The results of sequence analysis show that the full length of Gfa from Pseudomonas putida strain SG-32 is 393 bp; it encodes 131 amino acids. The gene was logged in GenBank, serial number is HM016790.BLAST search analysis showed that,Gfa gene from Pseudomonas putida strain SG-32 shares homology of 96% in nucleotide acid sequence compared with that of Pseudomonas putida F1.It has four regions, a VWFC domain , a C-terminal cystine knot , a Tubulin subunits alpha, beta and gamma signature and an Anaphylatoxin domain.The cloning of Gfa gene from Pseudomonas putida strain SG-32 provides a fundamentalbasis for further improvement of formaldehyde-resistanced of plants through genetic engineering.
1.抗甲醛细菌的分离与鉴定
分离了8株抗甲醛细菌,6个菌株能在20mmol/L甲醛的平板生长,命名为SG-32、SG-71、SG-87、SG-107、SG-122、SG-135。1个能在10mmol/L的甲醛平板上生长,命名为SG-zong。1个能在4mmol/L甲醛平板上生长,命名为SG-ju。经生理生化分析和分子鉴定,初步确定SG-32、SG-71、SG-87、SG-107、SG-122和SG-135是Pseudomonas putida恶臭假单胞菌。SG-zong是Pseudomonas fluorescens荧光假单胞菌。SG-ju是Acinetobacter johnsonii.约氏不动杆菌。
2.抗甲醛细菌生长特性和抗甲醛能力分析
SG-32、SG-71和SG-107最适生长温度为30℃,SG-135则是35℃。SG-32和SG-135的最适pH值为pH7。SG-71和SG-107的最适pH值为pH8。这4株菌可以耐受并代谢20 -35mmol/l的甲醛。
3.抗甲醛真菌的分离与鉴定
分离了3种抗甲醛真菌,SG-huang、SG-hei和SG-qing。通过形态学观察和分子鉴定,初步确定它们分别是Aspergillus nomius红绶曲霉、Aspergillus versicolor花斑曲霉和Penicillium vinaceum酒色青霉。
4.抗甲醛真菌的生长特性和抗甲醛能力分析
SG-huang、SG-he和SG-qing最适生长温度均为25℃。最适pH值为pH5。这3种真菌都可以降解甲醛,其中SG-huang7天内可降解80mmol/L甲醛,SG-hei7天内可降解50mmol/L甲醛,SG-qing7天内可降解30mmol/L甲醛。它们还能以甲醛作为唯一碳元进行代谢。
5.抗甲醛细菌Gfa基因的克隆
利用PCR法克隆了Pseudomonas putida strain SG-32的Gfa基因。该基因核苷酸序列全长为393bp,编码131个氨基酸。在GenBank进行了登录,序列号为HM016790。BLAST检索分析表明,Pseudomonas putida strain SG-32 Gfa基因与与Pseudomonas putida F1中依赖谷胱甘肽的甲醛激活酶(glutathione-dependent formaldehyde-activating,Gfa)序列同源性96%。该蛋白有4个区域,一个VWFC区,1个C-半胱氨酸节区,1个微管蛋白亚基α、β和γ识别区,1个过敏毒素区。Pseudomonas putida strain SG-32 Gfa基因的克隆,为今后利用该基因进行抗甲醛基因工程奠定了基础。
In this study, formaldehyde-resistanced bacterias and fungis were isolated from furniture factory sewage as the experimental material.The results were as follows.
1,Isolated and identified the formaldehyde-resistanced bacterias
8 strains formaldehyde-resistanced bacterias were isolated on selective media contaning formaldehyde.There are 6 isolated strains named as SG-32,SG-71,SG-87,SG-107,SG-122,SG-135 could grow on medium with 20mmol/L formaldehyde.One isolated strain named as SG-zong could grow on medium with 10mmol/L formaldehyde,and the anther one strain named as SG-ju could grow on medium with 4mmol/L formaldehyde.SG-32,SG-71,SG-87,SG-107,SG-122,SG-135 were identified as Pseudomonas putida by plentiful physiological-biochemical experiments and molecular identification.SG-zong was identified as Pseudomonas fluorescen.SG-ju was identified as Acinetobacter johnsonii.
2,Analysis the growth and the ability of formaldehyde-resistanced of bacterias
The optimal temperature were 30℃for SG-32,SG-71,SG-107 respectively and which were 35℃for SG-135. The optimal pH value were 7 for SG-32,SG-135 respectively and which were 8 for SG-71,SG-107.They all could tolerated 20mmol/L-35mmol/L formaldehyde.
3,Isolated and identified the formaldehyde-resistanced fungis
Formaldehyde-degrading fungis(SG-huang,SG-hei,SG-qing) were isolated and identified as Aspergillus nomius,Aspergillus versicolor,Penicillium vinaceum by morphological observation and molecular identification.
4,Analysis the growth and the ability of formaldehyde-resistanced of fungis
The optimal temperature were 25℃for SG-huang,SG-he,SG-qing respectively.The optimal pH value were 5 for SG-huang,SG-he,SG-qing respectively.After shaking culturing 7 days, formaldehyde degraded from 80, 50, 30mmol/L to almost 0 mmol/L bySG-huang,SG-he,SG-qing respectively.All the isolated strains could grow on medium with formaldehyde as the sole carbon source.
5,Cloning of Gfa from the formaldehyde-resistanced bacteria
The DNA sequence of Gfa gene from Pseudomonas putida strain SG-32 was determined by PCR.The results of sequence analysis show that the full length of Gfa from Pseudomonas putida strain SG-32 is 393 bp; it encodes 131 amino acids. The gene was logged in GenBank, serial number is HM016790.BLAST search analysis showed that,Gfa gene from Pseudomonas putida strain SG-32 shares homology of 96% in nucleotide acid sequence compared with that of Pseudomonas putida F1.It has four regions, a VWFC domain , a C-terminal cystine knot , a Tubulin subunits alpha, beta and gamma signature and an Anaphylatoxin domain.The cloning of Gfa gene from Pseudomonas putida strain SG-32 provides a fundamentalbasis for further improvement of formaldehyde-resistanced of plants through genetic engineering.
引文
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