摘要： 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2 727 bp。在已经构建条斑紫菜TPS基因原核表达载体pET22b-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。

关键词: 坛紫菜, TPS基因, 原核表达, 重组菌, 耐盐性

Abstract: 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 OD₆₀₀ 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.

Key words: Porphyra haitanensis, TPS gene, Prokaryotic expression, Recombinant E. coli, Salt tolerance

中图分类号: 

• S917.3