水曲柳FmPHV基因克隆及在形成层愈伤组织中的表达分析

Cloning and expression analysis of FmPHV gene in callus of Fraxinus mandshurica

关键词:  水曲柳, PHV基因, 生物信息学分析, 瞬时侵染, 形成层细胞

Key words:  Fraxinus mandshurica, PHV gene, bioinformatic analysis, transient infection, cambium cell

摘要:

该研究通过基因克隆获得了水曲柳PHV基因,并命名为FmPHV。生物信息学分析表明,水曲柳FmPHV基因编码区全长为2 112 bp,包含有一个完整的开放阅读框,其编码了一个由703个氨基酸组成的蛋白。亚细胞定位预测其主要存在于叶绿体中,为稳定亲水蛋白。保守域及同源分析表明,FmPHV与油橄榄、芝麻和烟草等物种的同源蛋白保守结构域同源性高达99%。在低温4 ℃条件下,使用50 mg·L^-1吲哚丁酸(IBA)溶液对水曲柳树皮进行处理,获得了水曲柳形成层细胞,并进一步诱导获得形成层愈伤组织。对FmPHV基因的时空表达模式进行分析表明,FmPHV基因6月表达量最高,同时FmPHV能在芽中高表达,通过树皮获得的不同来源的愈伤组织相互比较可知,FmPHV在来源为形成层分生组织形成层愈伤组织中的表达量显著高于其在其他来源的愈伤组织中的表达。此外,对水曲柳幼苗瞬时过表达FmPHV基因,对其所在通路关键基因的表达特征进行分析,FmPHV瞬时过表达后,生长素相关基因表达下降,细胞分裂素相关基因表达上升,有利于芽的分化。以上结果表明,揭示了水曲柳FmPHV在水曲柳植株生长过程中的表达模式以及FmPHV过表达对芽再生通路各关键基因的调控情况,为研究水曲柳FmPHV调控生长发育的分子机制以及其在生长素和细胞分裂素响应通路中发挥的作用奠定基础。

Abstract:

PHAVOLUTA(PHV)transcription factor is one of the most important members of homeodomain-leucine zipper Ⅲ(HD-ZIP)family, and plays an important role in the regulation of formation of plant stems apical and germ meristem. In this research, the PHV gene of Fraxinus mandshurica was acquired by gene cloning and was named by FmPHV. Detailed bioinformatics analysis showed that the length of FmPHV gene was 2 112 bp, and contained a complete Open Reading Frame(ORF)which encoded a protein with 703 amino acids. The FmPHV gene encoded a stable hydrophilic protein, the subcellular localization prediction of FmPHV protein showed the FmPHV was mainly present in the chloroplast. Conserved domain and homology analysis of FmPHV protein indicated that, the homology of FmPHV between the homology PHV proteins in other species, such as Olea europaean, Sesamum indicum and Nicotiana tabacum, was 99%. Under 4 ℃ condition, with the treatment of 50 mg·L^-1 3-indolebutyric acid(IBA)solution, we obtained the cambium callus from cambium stem cells of Fraxinus mandshurica. Analysis of the gene expression pattern of FmPHV showed that the FmPHV was the highest expression in June. Furthermore, the expression of FmPHV in buds was stronger than which in any other tissues. Compared with the callus from different sources, which obtained from the bark of F. mandshurica, the expression level of FmPHV gene in callus, derived from meristem formation, was significantly higher than which from other sources. Moreover, overexpression the FmPHV gene in F. mandshurica seedlings will reduce the expression of auxin-related genes and increase the expression of cytokinin-related genes. This phenomenon indicated that the function of FmPHV gene was more beneficial to the process of plant shoot differentiation and regeneration. In summary, this research revealed the expression pattern of FmPHV gene during the growth and development in F. mandshurica. And revealed the expression pattern of key genes in plant shoot regeneration pathway, which were regulated by FmPHV gene. Our research reveals the potential value of the molecular characteristic of FmPHV gene in the response of auxin and cytokinin pathways during the process of the formation of plant callus generation.