条斑紫菜TPS基因TPP结构域的缺失突变

摘要

Trehalose-6-phosphate synthase gene of Poryhyra yezoensis (PyTPS) contained both TPS and TPP domains.Using the prokaryotic expression vector of PyTPS gene (pET22b-PyTPS) as material, TPP domain of PyTPS gene was deleted through replacement of double digestion DNA fragment.First, the coding sequence Box1 of one phosphatase motif in TPP domain was deleted by replacement of an artificially synthesized 715bp KpnI-EcoRI DNA fragment, and Box1-deficiency mutation gene of PyTPS named PyTPS-Box1 was obtained.Second, coding sequence Box2 of another phosphatase motif in TPP domain was deleted by replacement of an artificial 661bp EcoRI-HindIII DNA fragment, and the yielding gene was Box2-deficiency mutation gene of PyTPS named PyTPS-Box2.Third, after deletion of Box1, Box2 was further deleted, and the mutation gene of PyTPS without both Box1 and Box2 named PyTPS-Box1-Box2) was finally yielded.Three mutated PyTPS genes and their prokaryotic expression vectors were obtained, and they were suitable materials for coding function research of PyTPS gene.%条斑紫菜6-磷酸海藻糖合成酶(TPS)基因(PyTPS)具有TPS及TPP双结构域,以该基因的原核表达载体pET22b-PyTPS为材料,利用双酶切片段替换法对该基因TPP结构域磷酸酶基序的编码框Box1与Box2进行了缺失突变.第一,用715bp缺失Box1的KpnI-EcoRI人工合成片段替换pET22b-PyTPS中相应的片段,获得了缺失Box1的突变基因PyTPS-Box1.第二,用661bp缺失Box2的EcoRI-HindII片段进行替换,获得了缺失Box2的突变基因PyTPS-Box2.第三,在缺失Box1后,再通过替换缺失Box2,获得了同时缺失Box1与Box2的突变基因PyTPS-Box1-Box2.本研究既获得了三种突变基因,又获得了其原核表达载体,为研究PyTPS基因的编码活性提供了材料,奠定了基础.