Shi J, Gonzales R A, Bhattacharyya M K
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Plant J. 1995 Sep;8(3):381-90. doi: 10.1046/j.1365-313x.1995.08030381.x.
Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian delta-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian delta-type PI-PLCs and yeast PI-PLC1, the putative Ca(2+)-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.
磷酸肌醇特异性磷脂酶C（PI-PLC）是一种关键的信号转导酶，可在哺乳动物细胞中生成第二信使三磷酸肌醇和二酰基甘油。通过使用抗（质膜）血清筛选cDNA表达文库，从大豆中分离出一个编码磷酸肌醇特异性磷脂酶C的cDNA克隆（PI-PLC1）。基因组DNA凝胶印迹分析表明，相应的基因是一个多基因家族的成员。大豆PI-PLC1同工酶推导的氨基酸序列包含在其他PI-PLC中发现的保守X和Y区域。就保守区域的排列而言，它与哺乳动物δ型PI-PLC、盘基网柄菌PI-PLC和酵母PI-PLC1密切相关。与哺乳动物δ型PI-PLC和酵母PI-PLC1不同，大豆PI-PLC1的假定Ca(2+)结合位点位于跨越X和Y结构域的区域，并且N末端区域被截断。从转基因烟草植物中纯化的带有FLAG表位标签的PI-PLC1融合蛋白显示出磷酸肌醇特异性磷脂酶C活性。大豆PI-PLC1 cDNA在酵母PI-PLC1缺失突变体中的异源表达补充了单倍体PI-PLC1破坏体的致死表型。对过表达带有FLAG表位标签的PI-PLC1融合蛋白的转基因烟草植物制备的细胞组分进行免疫印迹分析表明，PI-PLC1 cDNA编码的蛋白定位于细胞质和质膜中。
