林可链霉菌黑色素生物合成基因的克隆与表达

CLONING AND CHARACTERIZATION OF MELANIN BIOSYNTHESIS GENES FROM STREPTOMYCES LINCOLNENSIS 78-11

以pIJ702的melCl－C2基因为探针杂交林可链霉菌（Streptomyceslincolnensis）78－11染色体DNA，呈现出3．2kb的BamHI片段和2．6kb的SphI片段等一系列阳性条带。构建了含3．0～3．5kbBamHI片段的林可链霉菌78-11基因文库，从中分离克隆了黑色素生物合成基因melCl和melC2，并测定了含有mel基因的重组子pRSB336插入片段的全部DNA顺序。3152bpBamHI片段含有5个开放阅读框架，其中melCl和melC2与链霉菌属三个种的相应基因具有较高的同源性。此外，林可链霉菌78－11的melC2基因产物与人和鼠的酪氨酸酶轻微同源，分别为17．3％和24．5％。种种迹象表明，melCl、melC2和orf3组成黑色素生物合成操纵子结构。为了进一步鉴定上述克隆的林可链霉菌78-11黑色素生物合成基因，构建了分别含有新霉素抗性基因启动子和正反方向mel基因的重组质粒pPZ518和pPZ519，并转化变铅青链霉菌TK23。随机挑选的12个pPZ518转化子在R2YE培养基上均能分泌淡褐色色素，而所有的pPZ519和pES1转化子则都呈白色。

Using mel C1-C2 gene from pIJ702 as probe to hybridize wity chromosome DNA of S.lincolnensis 78-11,a series of positive bands were found.including a 3.2kb BanHI fragment and a 2.6kb SphI fragment. So a gene library of S.lincolnensis,comaing 3.0～3.5kb BamHI fragment,was constructed. Melanin biosynthesis genes,mel C1 and melC2,were then cloned and the fragment inserted in recombinant Plasmid pRSB336 were sequenced. Among five ORFs of the 3152bp BamHI fragment,mel C1 and melC2 were highly homologous to the respective genes of the other three stains of Streptomyces,morever,the product of mel C2 gene from S.lincolnensis 78-11 was slightly homologous to tyrosinase from human-being by 17.3% and from rat by 24.5%.All of the above results indicated that mel C1, mel C2 and orf 3 constitute the melanin biosynthesis operon of S.lincolnensis. In the further study of the cloned melanin biosynthesis genes of S.lincolnensis 78-11,recombinant plasmids,pPZ518 and pPZ519,which contain promoter of neo gene and mel gene in different orientation respectively, were constructed, and then were used to transform S. lividans TK23.12 transformants of pPZ518,selected at random,secreted light brown Pigment in R2YE culture medium, but all transformants of pPZ519 and pES1 were white.