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木质素是梨(Pyrus)石细胞的重要成分之一,木质素合成代谢对石细胞形成发育有着重要影响。肉桂酸4-羟化酶(cinnamate 4-hydroxylase,C4H,EC 1.14.13.11)是细胞色素单加氧酶超家族中CYP73A亚家族的一员,同时为木质素合成代谢中的一个关键酶。本研究从砀山酥梨(Pyrus bretschneideri cv.Danshan Su)果实中克隆出一条肉桂酸4-羟化酶基因的全长c DNA序列,命名为Pb C4H,Gen Bank登录号为:KF663548。Pb C4H全长1 764 bp,具有1 515 bp的ORF,编码504个氨基酸。Pb C4H与多个物种C4H基因编码的氨基酸序列具有较高的同源性,说明其在进化过程中较为保守。结构域分析表明,Pb C4H具有跨膜结构域和典型的细胞色素P450结构域特征。q RT-PCR分析发现在梨果实发育的不同时期,Pb C4H基因表达量呈现先增加后下降的趋势,其中在花后55 d达到最高。构建了Pb C4H原核表达载体p ET-32aPb C4H,在大肠杆菌(Escherichia coli)BL21菌株中诱导表达出Pb C4H融合蛋白。亚细胞定位表明Pb C4H蛋白定位于细胞膜上。本研究结果为利用分子生物学技术调控梨木质素的合成和石细胞的发育提供了基础资料。
Lignin is one of the most important components of Pyrus pebbles. Lignin anabolism plays an important role in the formation and development of stone cells. Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) is a member of the CYP73A subfamily of the cytochrome monooxygenase superfamily and a key enzyme in lignin anabolism. In this study, we cloned the full length c DNA sequence of cinnamic acid 4-hydroxylase gene from Pyrus bretschneideri cv. Danshan Su and named Pb C4H, Gen Bank accession number: KF663548. Pb C4H is 1 764 bp in length and has a 1 515 bp ORF encoding 504 amino acids. Pb C4H has high homology with the amino acid sequence encoded by C4H genes of many species, indicating that it is more conservative in the evolutionary process. Domain analysis showed that Pb C4H has the transmembrane domain and typical cytochrome P450 domain characteristics. q RT-PCR analysis showed that Pb C4H gene expression increased first and then decreased in different stages of pear fruit development, and reached the peak at 55 days after anthesis. The Pb C4H prokaryotic expression vector p ET-32aPb C4H was constructed and the Pb C4H fusion protein was induced in Escherichia coli BL21 strain. Subcellular localization indicates that the Pb C4H protein is localized on the cell membrane. The results of this study provide the basic information for the use of molecular biology techniques to regulate the synthesis of lignin and the development of stone cells.