偏肿革裥菌漆酶基因克隆及启动子序列分析
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摘要
以优化后的漆酶培养基为基础,通过RT-PCR和RACE技术相结合,从偏肿革裥菌Lenzites gibbosa菌株中获得编码漆酶基因的cDNA及Genomic DNA的全长序列,Genomic DNA大小为2 165 bp.通过比较该漆酶基因的cDNA和Genomic DNA的全长序列,发现该基因包含11个外显子和10个内含子.cDNA序列的全长为1 873 bp,其中包含一个完整的ORF,长度为1 563 bp,编码520个氨基酸.序列在氨基酸水平上与彩绒革盖菌Trametes versicolor的相似性评价最高,相似性达83%.通过SEFA-PCR的方法,扩增得到漆酶基因起始密码子上游长986 bp的启动子序列.分析表明,该启动子区域上除分布有TATA-box、CAAT-box以及AP2等基本的转录起始元件外,还存在有多个潜在的顺式作用元件序列位点,包括7个MRE元件、2个STRE元件、1个HSEs元件、7个氮因子结合位点等.这些结果表明,不同的外源诱导物可以调节偏肿革裥菌漆酶基因的表达.
The cDNA and Genomic DNA sequences of the laccase gene from Lenzites gibbosa were obtained by PCR and RACE technology,and the length of laccase gene was 2 165 bp. Comparison of the cDNA and DNA sequences showed that the laccase gene contained 11 exons and 10 introns. The cDNA length of laccase gene was 1 873 bp,including an ORF with 1 563 bp length and codes 520 amino acids.The closest organism was Trametes versicolor with 83% similarity level of amino acids. The 986 bp sequences of promoter located in the upstream of start code of laccase gene were obtained by the approach of SEFA-PCR. The promoter not only scattered in the basic transcriptional elements of TATA-box,CAATbox and AP2,but also contained seven elements of MRE,two elements of STRE,one element of HSEs and seven nitrogen factor binding sites,etc. The laccase gene expression of the Lenzites gibbosa can be regulated by different exterior inducers.
The cDNA and Genomic DNA sequences of the laccase gene from Lenzites gibbosa were obtained by PCR and RACE technology,and the length of laccase gene was 2 165 bp. Comparison of the cDNA and DNA sequences showed that the laccase gene contained 11 exons and 10 introns. The cDNA length of laccase gene was 1 873 bp,including an ORF with 1 563 bp length and codes 520 amino acids.The closest organism was Trametes versicolor with 83% similarity level of amino acids. The 986 bp sequences of promoter located in the upstream of start code of laccase gene were obtained by the approach of SEFA-PCR. The promoter not only scattered in the basic transcriptional elements of TATA-box,CAATbox and AP2,but also contained seven elements of MRE,two elements of STRE,one element of HSEs and seven nitrogen factor binding sites,etc. The laccase gene expression of the Lenzites gibbosa can be regulated by different exterior inducers.
引文
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[11]MOLDES D,SANROMN M.Amelioration of the ability to decolorize dyes by laccase:Relationship between redox mediators and laccase isoenzymes in Trametes versicolor[J].World J Microbiol Biotechnol,2006,22(11):1197-1204.
[12]TONG P,HONG Y,XIAO Y,et al.High production of laccase by a new basidiomycete,Trametes sp.[J].Biotechnol Lett,2007,29(2):295-301.
[2]FARACO V,GIARDINA P,SANNIA G.Metal-responsive elements in Pleurotus ostreatus laccase gene promoters[J].Microbiology,2003,149(8):2155-2162.
[3]RUSHMORE T H,KING R G,PAULSON K E.Regulation of glutathione S-transferase Ya subunit gene expression:Identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds[J].Proc Natl Acad Sci USA,1990,87(10):3826-3830.
[4]SODEN D M,DOBSON A D W.Differential regulation of laccase gene expression in Pleurotus sajor-caju[J].Microbiology,2001,147(7):1755-1763.
[5]XIAO Y Z,CHEN Q,HANG J,et al.Selective induction,purification and characterization of a laccase isozyme from the basidiomycete Trametes sp.AH28-2[J].Mycologia,2004,96(1):26-35.
[6]MARZLUF G A.Genetic regulation of nitrogen metabolism in the fungi[J].Microbiol Mol Biol Rev,1997,61(1):17-32.
[7]陈军,高大文,池玉杰,等.偏肿拟栓菌Pseudotrametes gibbosa产漆酶的条件优化[J].菌物学报,2008,27(6):940-946.
[8]LIU Yaoguang,CHEN Yuanling.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques,2007,43(5):649-50.
[9]TREGER J M,MAGEE T R,McENTEE K.Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae[J].Biochem Biophys Res Commun,1998,243(1):13-19.
[10]STRAUSS J,HORVATH H K,ABDALLAH B M,et al.The function of CreA,the carbon catabolite repressor of Aspergillus nidulans,is regulated at the transcriptiosl and post-transcriptional level[J].Molecular microbiology,1999,32(1):169-178.
[11]MOLDES D,SANROMN M.Amelioration of the ability to decolorize dyes by laccase:Relationship between redox mediators and laccase isoenzymes in Trametes versicolor[J].World J Microbiol Biotechnol,2006,22(11):1197-1204.
[12]TONG P,HONG Y,XIAO Y,et al.High production of laccase by a new basidiomycete,Trametes sp.[J].Biotechnol Lett,2007,29(2):295-301.