假单胞菌中聚羟基脂肪酸酯合成酶基因的克隆与分析

Cloning and Analysis of Genes Encoding Polyhydroxyalkanoate Synthases

大多数二型聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHA)合成酶(PhaC)发现于假单胞菌中,其基因组成形式为两个PhaC基因中间有一个PHA降解酶PhaZ基因.根据已知的假单胞菌PHA合成酶基因区域的特殊结构,设计了采用PCR方法从假单胞菌中克隆PHA合成酶基因的克隆方案,并成功地对一株硝基还原假单胞菌(Pseudomonasnitroreducens)和一株石竹伯克霍尔德氏菌(Burkholderiacaryophylli)中的PHA合成酶基因进行了克隆.将克隆得到的phaC1,phaZ和phaC23个基因所编码的氨基酸序列与其他6株已知PHA合成酶基因的假单胞菌的相应序列进行比较,发现这3个蛋白质在假单胞菌中的相似性很高,由phaC1,phaZ和phaC23个基因所组成的基因区域在假单胞菌中非常保守.从对PhaC1,PhaZ和PhaC2的系统进化树分析可以发现,这3个蛋白质与整个PHA合成酶基因区域的系统进化树有着一致的结构.这表明假单胞菌中的PHA合成酶基因区域可能起源于共同的祖先,而其形成可能经历了一次PHA合成酶基因加倍的过程.

Most type II polyhydroxyalkanoate (PHA) synthases (PhaC) were found in Pseudomonas strains, and their genes are colocalized with PHA depolymerase (PhaZ) genes. Recently, two similar PHA biosynthesis gene clusters were cloned and analyzed from Pseudomonas nitroreducens and Burkholderia caryophylli in our lab. The deduced amino acid sequences of the three genes (phaC1, phaZ and phaC2) in the clusters were compared with those from six other Pseudomonas strains (Pseudomonas sp. 613, P. aeruginosa, P. aureofaciens, P. oleovorans, P. putida and P. resinovorans) containing the homologous pha clusters. The results showed that the three proteins PhaC1, PhaZ and PhaC2 shared high similarity. The pha clusters were conserved in the eight strains. The phylogenetic trees for PhaC1, PhaZ and PhaC2 of the eight strains were compared and same topology as that of the whole pha clusters (phaC1phaZphaC2 DNA sequence) from the eight strains was found. This result implied that the pha cluster in Pseudomonas spp. may arise from same ancestor. An ancient duplication event of PHA synthase genes to form the pha cluster may happen earlier than Pseudomonas strains. A hypothetical evolution history was proposed based on the analysis.