CAJ | 학술논문

以叶状体总 DNA 为模板，用3对DNA 引物进行 PCR 扩增，获得了坛紫菜6-磷酸海藻糖合成酶基因（PhTPS）3个重叠的片段，大小分别为1．3，1．1，0．7 kb。用pMD18-T载体克隆这些片段，经测序与序列拼接获得了该基因完整的ORF序列，其长度为2727 bp。在已经构建条斑紫菜TPS基因原核表达载体pET22 b-PyTPS的基础上，用PhTPS基因替代该表达载体中的PyTPS基因，获得了 PhTPS基因的原核表达载体 pET22b-PhTPS。重组菌BL21（pET22b-PhTPS）经IPTG诱导，SDS-PAGE显示获得了约100 kDa的特异性蛋白条带；目测及OD600的测定结果表明重组菌的耐盐性明显高于对照菌，即PhTPS基因的表达提高了重组菌的耐盐性。为利用PhTPS基因进行作物耐盐等抗逆转基因改良提供了依据。
이협상체총 DNA 위모판，용3대DNA 인물진행 PCR 확증，획득료단자채6-린산해조당합성매기인（PhTPS）3개중첩적편단，대소분별위1．3，1．1，0．7 kb。용pMD18-T재체극륭저사편단，경측서여서렬병접획득료해기인완정적ORF서렬，기장도위2727 bp。재이경구건조반자채TPS기인원핵표체재체pET22 b-PyTPS적기출상，용PhTPS기인체대해표체재체중적PyTPS기인，획득료 PhTPS기인적원핵표체재체 pET22b-PhTPS。중조균BL21（pET22b-PhTPS）경IPTG유도，SDS-PAGE현시획득료약100 kDa적특이성단백조대；목측급OD600적측정결과표명중조균적내염성명현고우대조균，즉PhTPS기인적표체제고료중조균적내염성。위이용PhTPS기인진행작물내염등항역전기인개량제공료의거。
Using total DNA of Porphyra haitanensis thalli as PCR template,three overlapping fragments of its trehalose-6-phosphate synthase(TPS)gene(PhTPS)with size of 1.3,1.1,0.7 kb were amplified by three pairs of DNA primers respectively .After T-A cloning ,sequencing and sequence assembly ,the full length open reading frame sequence of PhTPS gene was obtained.Without intron,its length is 2 727 bp.Based on the construction of the pro-karyotic expression vector of Porphyra yezoensis TPS gene ( pET22 b-PyTPS ) , its PyTPS gene was replaced by PhTPS gene and the prokaryotic expression vector of PhTPS gene(pET22b-PhTPS)was derived.After expression induction of the recombinant E.coli strain BL21(pET22b-PhTPS)with TPTG,a specific band about 100 kDa was found in SDS-PAGE pattern.Salt tolerance of the recombinant strain was detected in the NaCl-containing LB medi-um,visual inspection and OD 600 determination results indicated that the salt-tolerance of the recombinant strain was significantly higher than that of control E.coli strain BL21(pET22b).The salt tolerance of the recombinant strain was increased by the expression of PhTPS gene.This gene can be used as a salt-tolerant candidate gene in plant ge-netic engineering improvement .