香菇顺式调控元件的克隆及其序列分析
CLONING AND SEQUENCE ANALYSIS OF CIS-ACTING ELEMENT OF LENTINULA EDODES
胡乐琴 姚泉洪 陈明杰 熊爱生 潘迎捷
摘 要：应用顺式调控元件探测载体G221构建了一个香菇Lentinula edodes基因组文库.G221为大肠杆菌-酿酒酵母穿梭载体,含有一个由酵母Cyc1基因基本启动子控制的lacZ标记基因,能以转录增强活性筛选香菇DNA片段.用这个基因组文库转化酵母菌,获得了一批lacZ阳性转化子.对其中表达较强的阳性转化子进行质粒抽提和双酶切鉴定,筛选到50个香菇顺式调控元件DNA片段.对其中部分片段进行了测序,并对其中一个序列进行了序列分析,鉴别了该序列上的几个与转录相关的特征序列.该研究也探讨了利用酵母表达系统克隆香菇顺式调控元件的可行性.
关键词：食用菌; 基因组文库; 转录增强活性; LacZ
分类号：Q939.96 文献标识码：A
文章编号：1007-3515(2002)03-0406-0411
基金项目：本研究由上海科技兴农重点攻关项目资金资助
作者简介：胡乐琴:huyueqin@yahoo.com,通讯作者潘迎捷
作者单位：胡乐琴（南京农业大学资环学院,南京,210095）
姚泉洪（上海农科院生物技术研究中心,上海,201106）
陈明杰（上海农科院食用菌研究所,农业部食用菌遗传育种重点开放实验室,上海,201106）
熊爱生（上海农科院生物技术研究中心,上海,201106）
潘迎捷（上海农科院食用菌研究所,农业部食用菌遗传育种重点开放实验室,上海,201106）
参考文献：
［1］Chen X, Stone M, Schlagnhaufer C et al.,2000. A fruiting body tissue method for efficient Agrobacterium --mediated transformation of Agaricus bisporus. Applied Environ Microbiol, 66: 4510～4513
［2］Dower SJ, Miller JF, Ragsdale CW,1988. High efficiency transformation of E.coli by high voltage electroporation. Nucleic Acids Research, 16(13): 6127～6145
［3］Froeliger EH,Ullrich RC,Novotny CP,1989. Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune. Gene, 83(2): 387～393
［4］Gietz D, Jean AS, Woods Ra et al.,1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res, 20: 1425
［5］Hirano T, T.Sato T, Yaegashi K et al., 2000. Efficient transformation of the edible basidiomycete Lentinula edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance.Mol Gen Genet, 263: 1047～1052
［6］Jia JH, Buswell JA, Peberdy J,1998. Transformation of the edible fungi ,Pleurotus ostreatus and Volvariella volvacea. Mycol Res,102 (7): 876～880
［7］Mooibroek H, Kuipers AGJ, Sietsma JH et al.,1990. Introduction of hygromycin B resistance into Schizophyllum commune :Preferential methylation of donor DNA. Mol Gen Genet, 222: 41～48.
［8］Robzyk K, Kassir Y,1992. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucl Acids Res, 20: 3790
［9］Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Manual(2nd Ed ) Cold Spring Harbor, NY : Cold Spring Harbor,Laboratory Press. 1～69
［10］Santos C, Labarere J, 1999. Aa-Pri2, a single-copy gene from Agrocybe aegerita, specifically expressed during fruiting initiation, encodes a hydrophobin with a leucine-zipper domain. Curr Genet, 35(5): 564～70
［11］Sato T,Yaegashi K,Ishii S et al., Transformation of the Edible basidiomycete Lentinula edodes by restriction enzyme-mediated integration of plasmid DNA. Biosci Biotechn. Biochem, 62 : 2346～2350
［12］Sherman F,1991. Getting started with yeast. Methods Enzymol, 194: 3～21
［13］Zhang YZ, Reddy CA, Rasooly A, 1991. Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium. Gene, 97(2): 191～198
