Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler)
Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler)
复制标题
DOI：
10.1093/jxb/erf029
复制
发表时间：
2002-08-01
影响因子：
6.9
通讯作者：
Muñoz-Blanco, J
中科院分区：
文献类型：
--
作者：
Blanco-Portales, R;Medina-Escobar, N;Muñoz-Blanco, J
肉桂醇脱氢酶（CAD; EC 1.1.1.195）催化对羟基肉桂醛转化为相应的醇，并且被认为是木质素生物合成中的关键酶。通过草莓（Fragariax ananassa cv.钱德勒）果实特异性消减cDNA文库中，分离出对应于cad基因的全长克隆（Fxacad 1）。北方印迹和定量真实的时间PCR研究表明，草莓Fxacad 1基因表达的果实，亚军，叶，花，但不是在根。此外，该基因在果实沿着成熟过程中呈现差异表达。此外，通过筛选草莓基因组文库，分离出cad基因（Fxacad 2）。与其他来自高等植物的cad基因相似，草莓cad基因由5个外显子和4个内含子组成。Southern杂交分析表明，草莓中可能存在一个小的cad基因家族。RT-PCR研究表明，只有Fxacad 1基因表达在所有的果实成熟阶段和营养组织分析。Fxacad 1 cDNA在E.大肠杆菌细胞，并使用相应的蛋白质来产生针对草莓CAD多肽的抗体。获得的抗体用于免疫定位研究。结果表明，CAD多肽定位于所有组织（瘦果，果托，流道，叶，花梗和花）的木质化细胞。此外，cDNA也在酵母（毕赤酵母）中作为胞外蛋白表达。重组蛋白对被子植物CAD酶的特征底物具有活性，表明克隆的基因对应于CAD蛋白。
Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1). Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2). Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry. RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The Fxacad1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.