Villalobos E, Santos M, Talavera D, Rodríguez-Falcón M, Torné J M
Departament de Genètica Molecular, Institut de Biología Molecular de Barcelona, Cordi Girona 18-26, 08034 Barcelona, Spain.
Gene. 2004 Jul 7;336(1):93-104. doi: 10.1016/j.gene.2004.03.025.
Two related complementary DNA clones, TGZ15 and TGZ21, encoding active maize transglutaminase (TGase) have been isolated for the first time in plants by molecular cloning (Patent Pending PCT/ES03/00247). Southern and northern blot analyses indicate that the two cDNAs probably corresponded to two different single-copy genes in the maize genome. Northern blot analyses revealed that the transcript is expressed preferentially in young leaves and differentiated embryogenic maize callus. This expression is dependent on light exposure time. TGase activity of the proteins encoded by clones TGZ15 and TGZ21 was detected in bacterial extracts overexpressing them, using two enzymatic assays. TGase activity was significantly higher than that of the empty-phagemid bacterial extracts. As in other TGases, this activity was inhibited by monodansyl cadaverine (MDC), GTP and the absence of exogenous Ca(2+). Likewise, light-stimulated Ca(2+)-dependent TGase activity was detected in thylakoids and grana of maize chloroplast, which was inhibited by MDC, GTP, DIECA and Diuron.
通过分子克隆首次在植物中分离出两个相关的互补DNA克隆TGZ15和TGZ21，它们编码活性玉米转谷氨酰胺酶（TGase）（专利申请中，PCT/ES03/00247）。Southern和Northern印迹分析表明，这两个cDNA可能对应于玉米基因组中的两个不同单拷贝基因。Northern印迹分析显示，该转录本在幼叶和分化的胚性玉米愈伤组织中优先表达。这种表达取决于光照时间。使用两种酶促测定法在过表达克隆TGZ15和TGZ21的细菌提取物中检测到了它们所编码蛋白质的TGase活性。TGase活性明显高于空噬菌粒细菌提取物。与其他TGase一样，这种活性受到单丹磺酰尸胺（MDC）、GTP和缺乏外源Ca(2+)的抑制。同样，在玉米叶绿体的类囊体和基粒中检测到了光刺激的Ca(2+)依赖性TGase活性，该活性受到MDC、GTP、二乙基二硫代氨基甲酸钠（DIECA）和敌草隆的抑制。
