chiA基因在稻根联合固氮菌E26和NG13中的表达

EXPRESSION OF THE CHIA GENE FROM SERRATIA MARCESCENS IN BOTH STRAIN E26 AND STRAIN NG13, NITROGEN-FIXING BACTERIA ASSOCIATED WITH RICE ROOT

将带有粘质沙雷氏菌几丁质酶基因 (chiA)的 1 8kbHinfⅠ片段分别克隆到表达载体pKK2 2 3 3和质粒pMC71A上 ,构建成几丁质酶表达质粒pKChiA和pMChiA。将这 2种质粒转化稻根联合固氮菌阴沟肠杆菌E2 6 (EnterobactercloacaeE2 6 )和催娩克氏菌NG1 3 (Klebsiellaoxy tocaNG1 3 ) ,chiA基因在这 2菌株中获得高效表达。对表达产物的细胞定位测定表明 ,几丁质酶不仅存在于细胞周间质和胞内 ,而且还分泌到培养物上清液中。在对数生长后期 ,胞外、胞间质和胞内的几丁质酶活性分布分别为 2 3 %～ 2 8%、45 %～ 5 1 %和 2 1 %～ 3 2 %。经SDS 聚丙烯酰胺凝胶电泳检测表明 ,表达的几丁质酶蛋白分子量为 5 8kD。在受体细胞内 ,质粒pMChiA的稳定性要比质粒pKChiA高。

A 1.8kb HinfI fragment carrying the chitinase gene (chiA) from Serratia marcescens was cloned into the expression vector pKK223-3 and the plasmid pMC71A,yielded the plasmid pKChiA and the plasmid pMChiA respectively.Both plasmid pKChiA and plasmid pMChiA were used to transform to the Enterobacter cloacae strain E26 and the Klebsiella oxytoca strain NG13,two nitrogen-fixing bacteria associated with rice root.The chiA gene could be highly expressed in the ChiA++ transformants of the strain E26 or the strain NG13. Cell location determination of the expressed chitinase showed that the enzyme existed not only in cell periplasm and cytoplasm, but also in extracellular broth.When the cultures were in the aftor logarithmic growthe phase, the distribution of the enzyme activity in extracelluar broth, periplasm and cytoplasm were 23%～28%, 45%～51% and 21%～32%,respectively. The molecular weight of chitinase expressed in the ChiA++ transformants was 58kD by SDS-PAGE analysis.The stability of the plasmid pMChiA in the transformants was better than that of the plasmid pKChiA.