多聚磷酸盐激酶(PPK)基因植物表达载体的构建Construction of Plant-based Expression Vector of Polyphosphate Kinase Gene
曹访;杨志红;韩志萍;杨倩;费佳玲;
摘要(Abstract):
[目的]构建融合表达PPK和绿色荧光蛋白的融合表达载体pCAMBIA1302-PPK。[方法]根据GenBank中登录的大肠杆菌PPK基因序列(L03719)设计引物,以E.coli DH5α基因组DNA为模板,通过PCR扩增得PPK基因,然后用In-Fusion@HD Cloning Kit将PPK基因克隆到pCAMBIA1302载体的Nco I酶切位点。[结果]序列测定结果显示,pCAMBIA1302-PPK含有约2.0 kb的PPK基因片段,说明PPK基因已插入植物表达载体pCAMBIA1302的绿色荧光蛋白基因前。[结论]成功构建了融合表达PPK和绿色荧光蛋白的融合表达载体pCAMBIA1302-PPK。
关键词(KeyWords): 大肠杆菌;多聚磷酸盐激酶基因;植物表达载体;构建
基金项目(Foundation): 国家自然科学基金(31070451);; 浙江省钱江人才计划项目(2009R10016);; 浙江省自然科学基金(Y5110057)
作者(Author): 曹访;杨志红;韩志萍;杨倩;费佳玲;
Email:
DOI: 10.13989/j.cnki.0517-6611.2012.31.003
参考文献(References):
- [1]STEPHEN J,DIEN V,KEASLING J D.Control of polyphosphate metabo-lism in genetically engineered Escherichia coli[J].Enzyme and MicrobialTechnology,1999,24(1/2):21-25.
- [2]OGAWA N,TZENG C M,FRALEY C D,et al.Inorganic polyphosphate invibrio cholerae:genetic,biochemical,and physiologic features[J].Journalof Bacteriology,2000,182(23):6687-6693.
- [3]GAVIGAN J A,MARSHALL L M,DOBSON A W.Regulation of polyphos-phate kinase gene expression in Acinetobacter baumannii 252[J].Microbi-ology,1999,145:2931-2937.
- [4]王勤,赵庆顺,肖琳,等.转聚磷激酶基因的大肠杆菌去除水体中的磷[J].中国环境科学,2006,26(6):742-745.
- [5]管莉菠,蔡天明,李波,等.Pseudomonas putida GM6多聚磷酸盐激酶(ppk)基因的克隆及表达[J].土壤学报,2007,44(4):727-733.
- [6]ARCHIBALD F S,FRIDOVICH I.Invest igations of the st ate of the man-ganese in Lactobacillus plantarum[J].Arch Biochem Biophys,1982,215:589-596.
- [7]PAN HOU H,KIYONO M,KAWASE T,et al.Evaluation of ppk specifiedpolyphosphate as a mercury remedial tool[J].Biol Pharm Bull,2001,24:1423-1426.
- [8]PAN HOU H,KIYONO M,OMURA H,et al.Polyphosphat e produced inrecombinant Escherichia coli confers mercury resistance[J].FEMS Micro-biol Lett,2002,207:159-164.
- [9]TAKESHI N,MASAKO K,HIDEMITSU P.Engineering expression of bac-terial polyphosphate kinase in tobacco for mercury remediation[J].ApplMicrobiol Biot echnol,2006,72:777-782.
- [10]BIZILY S P,PUGH C L,SUMMERS A O,et al.Phytoremediation of meth-ylmercury pollution:merB expression in Arabidopsis thaliana confers resistance to organomercurials[J].Proc Natl Acad Sci USA,1999,96:6808-6813.
- [11]BIZILY S P,PUGH C L,MEAGHER R B.Phytodetoxification of hazard-ous organomercurials by genetically engineered plants[J].Nat Biotechn-ol,2000,18:213-217.
- [12]韩小艳,赵娜,王永祥.接头序列及其在融合蛋白构建中的应用[J].河北医科大学学报,2007,28(3):224-227.
- [13]杜宏伟,武俊,肖琳,等.聚磷激酶基因在假单胞菌中的整合和表达[J].环境科学,2009,30(10):3011-3015.
- [14]任晶,肖琳,郑小红,等.转聚磷激酶大肠杆菌诱导表达条件及磷浓度对其除磷能力的影响[J].环境保护科学,2008,34(6):1-4.