红海榄肉桂酸-4-羟基化酶基因的克隆与表达分析

Cloning and expression analysis of Cinnamate-4-hydroxylase gene from Rhizophora stylosa

肉桂酸-4-羟基化酶是木质素合成反应中的关键酶,木质素合成反应对于植物耐受重金属胁迫有重要作用。本研究利用同源克隆和cDNA末端快速扩增方法(rapid amplification of cDNA ends,RACE)从红海榄叶片中克隆得到一条肉桂酸-4-羟基化酶基因,命名为RsC4H,生物信息学分析结果显示RsC4H基因的cDNA全长为2006bp,开放阅读框长1572bp,共编码523个氨基酸。该基因编码蛋白的相对分子质量为60.18kD,是亲水性的不稳定蛋白,二级结构以α螺旋(47.42%)和无规则卷曲(32.70%)为主,该蛋白在N端和C端分别包含一个跨膜结构,不含有信号肽,主要在膜结构或内质网上发挥功能,属于p450超家族。RsC4H蛋白与其他植物的C4H蛋白相似性较高,系统发育树结果显示其与笋瓜(Cucurbita maxima, XP_023007159.1)、南瓜(Cucurbita moschata, XP_022947682.1)的亲缘关系更近。qRT-PCR结果显示,红海榄能快速响应重金属胁迫,提高RsC4H基因的表达量,促进木质素生成并增加细胞壁厚度以阻止金属离子进入细胞。此研究结果丰富了红树植物抗重金属胁迫基因库,为从分子水平揭示红海榄耐受重金属胁迫机制提供了基础资料。

Cinnamate-4-hydroxylase is a key enzyme in lignin synthesis which plays an important role in plant tolerance to heavy metal stress. In this study, a cinnamate-4-hydroxylase gene named RsC4H was cloned from the leaves of Rhizophora stylosa by homologous cloning and rapid amplification of cDNA ends(RACE). Bioinformatics analysis showed that the full length of RsC4H gene cDNA was 2006bp, the length of open reading frame was 1572bp, and encoded 523 amino acids. The relative molecular weight of the encoded protein is 60.18kD. It is a hydrophilic unstable protein with secondary structure alpha-helix(47.42%) and random coil(32.70%), this protein contains a transmembrane structure at the N-terminal and C-terminal, respectively, and does not contain signal peptides. It is mainly distributed on the membrane structure or endoplasmic reticulum to function, and belongs to P450 superfamily. RsC4H protein has high similarity with C4H protein of other plants. The phylogenetic tree results showed that it was closer to Cucurbita maxima(xp_023007159.1) and Cucurbita moschata(xp_022947682.1). qRT-PCR results showed that Rhizophora stylosa could quickly respond to heavy metal stress, improve the expression of RsC4H gene, promote lignin production, increase cell wall thickness and prevent metal ions from entering cells.The results enrich the gene pool of mangrove plants resistant to heavy metal stress, and lay a foundation for revealing the mechanism of heavy metal stress tolerance of mangrove at the molecular level.