马尾松细胞分裂素羟化酶基因PmCYP735A克隆与表达分析

Cloning and expression analysis of cytokinin hybroxylase gene PmCYP735A in Pinus massoniana

关键词:  细胞分裂素羟化酶, RACE, 表达模式, 马尾松, 系统进化分析

Key words:  cytokinin hydroxylase, RACE, expression pattern, Pinus massoniana, phylogenetic analysis

摘要:

该文首次在针叶树种马尾松(Pinus massoniana)中采用cDNA末端快速克隆(RACE)技术克隆,并鉴定出1个CYP735A基因。结果表明:马尾松CYP735A基因(PmCYP735A)cDNA全长为1 744 bp,包括1 647 bp的开放阅读框,44 bp的5'端非翻译区和53 bp的3'端非翻译区。该基因编码蛋白由548个氨基酸残基组成,其二级结构含有丰富的α-螺旋和无规则卷曲。该基因编码蛋白不含跨膜区域,且无信号肽酶切位点,在399～406个和475～484个氨基酸残基存在P450超家族保守特征序列ETLRLYP(ExxRxxP)和血红素结合区域(Heme-binding region)FSFGPRKCVG(FxxGxRxCxG)。系统进化树分析表明,马尾松PmCYP735A与水稻、玉米、拟南芥CYP735A蛋白归属于同一小的进化枝,可可、毛果杨、麻风树和橡胶树等的CYP735A同源蛋白相对集中的定位于另一进化分支。实时定量PCR检测发现,PmCYP735A基因在马尾松根和茎中的表达量显著高于叶,该基因响应外源生长素NAA诱导表达,随着诱导时间呈现先上升再下降的表达趋势。以上结果有助于深入探究CYP735A基因家族的生物学功能及其在物种间的表达调控异同,为进一步挖掘马尾松优异基因资源奠定基础。

Abstract:

Cytokinin is a sort of adenine derivatives that can promote cell division and differentiation, which is widely involved in plant organ morphogenesis and stress response. Cytokinin hydroxylase CYP735A belongs to the cytochrome P450 monooxygenase superfamily. It regulates the content of isoprenyl adenine and trans-zeatin in plants through hydroxylation reaction and plays an important role in plant growth and development. Up to now, there were few researches about gene cloning and analysis of cytokinin hydroxylase in woody plants. In this study, one CYP735A homologous gene was firstly cloned and identified in Pinus massoniana through the rapid amplification of cDNA ends(RACE). The results were as follows: The full-length of cDNA of PmCYP735A was 1 744 nucleotides, including 1 647 bp open reading frame(ORF), 44 bp 5'-untranslated region(UTR)and 53 bp 3'-UTR. PmCYP735A encoded a protein of 548 amino acids, and its secondary structure was rich in alpha helix and random coil. PmCYP735A protein did not contain either transmembrane region or signal peptidase cleavage site. But two conserved motifs of P450 superfamily such as ETLRLYP(ExxRxxP)and heme-binding region FSFGPRKCVG(FxxGxRxCxG)were existed in amino acid sequence of 399-406 aa and 475-484 aa, respectively. The phylogenetic tree showed that PmCYP735A was clustered into the same evolutionary branch with homologous of Arabidopsis thaliana, Oryza sativa and Zea mays, while CYP735A homologous proteins from Theobroma cacao, Populus trichocarpa and Jatropha curcas were classified in another evolutionary branch. The results of qRT-PCR showed that PmCYP735A expressed significantly higher in roots and stems than in leaves. Furthermore, the gene responded to exogenous auxin NAA with a increased firstly and then decreased gene expression trend over treatment time in all three tissues. These results will be valuable for further study of the biological function of CYP735A homologous genes in woody plants and enrich the excellent genetic resources of Pinus massoniana.