摘要
从土壤中分离到一株降解 2 ,4 二氯酚能力较强的细菌菌株GT2 41 1,经鉴定该菌株属于假单胞菌属。菌株GT2 41 1在最适条件下能在 48h内将 90mg L的 2 ,4 DCP降解 91% ,能利用 2 ,4 二氯酚、2 ,4 二氯苯氧乙酸、苯甲酸和儿茶酚为唯一碳源生长。采用Southern杂交对 2 ,4 二氯酚羟化酶基因 (dcpA)定位后构建基因组文库 ,再用斑点杂交筛选目的转化子 ,克隆了该菌株的dcpA。序列测定得知含dcpA的亚克隆片段全长 2 3 89bp ,其中dcpA基因编码区 1797bp。核苷酸和氨基酸序列分析表明 ,dcpA与已在GenBank登记的相关基因有一定的差异。
Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichloro phenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., w a s isolated from soil samples which was collected from drainage area of several 2 ,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichloro phenol degrading ability, it could decompose 91% 2,4-dichlorophenol of 90mg/L w ithin 48 hours at 25～30℃, and could utilize 2,4-dichlorophenol, 2,4-dichloro p henoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy sour ce. Southern blot showed that 2,4-dichlorophenol hydroxylase gene(dcpA) of strain GT241-1 locates on the about 10kb EcoRⅠ/XbaⅠ fra gment. This f ragment was recovered, linked to the vecter pUC19 and transformed into the E.c oli DH5α. A aim transformant, Z539, was obtained by dot blotting from about 1 200 transformants. PCR and the sequencing results shew that the whole dcpA gene i s contained within the 10kb EcoRⅠ/XbaⅠ fragment of pZ539 This f ragment was shortened to about 2 4kb by HindⅢ. The shorted fragment was su bcloned to vecter pRSET-B to get a transformant BS1-12 The subcloned fragmen t was sequencd. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA , this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transforman t Z539 and BS1-12 were detected, the results showed these transformants have 2, 4-dichlorophenol hydroxylase activity. By comparison, the activity of these tra nsformants were lower than that of the strain GT241-1.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第2期209-214,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目 (No .30 0 80 0 1 3)
教育部农业微生物重点开放实验室开放基金资助项目~~
