马尾松紫色酸性磷酸酶基因PmPAP1的克隆与表达模式分析

[目的]研究马尾松紫色酸性磷酸酶基因功能及其对低磷胁迫的应答。[方法]采用RACE法克隆马尾松紫色酸性磷酸酶(PAPs)家族成员Pm PAP1,利用软件和数据库,对基因结构功能进行多重分析预测,检测其接种外生真菌及低磷胁迫下的表达模式。[结果]表明,Pm PAP1基因c DNA全长2 520 bp,开放阅读框1 869 bp,编码622个氨基酸残基,具紫色酸性磷酸酶保守结构域特征,属于高分子量PAPs。Pm PAP1有信号肽,无跨膜区,推测定位于细胞质基质或细胞器基质中,它与莲(Nelumbo nucifera)、无油樟(Amborella trichopoda)的亲缘关系最近,相似性分别达71%和69%,进化关系古老、保守性强。时空表达分析表明,Pm PAP1的表达受外生菌根诱导,在不同组织中均有表达,其根中的表达量显著高于茎和叶。在菌根化和非菌根化的幼苗中,Pm PAP1的表达均受基质中磷含量的影响,表现为低磷条件下高效激活,高磷条件下其表达反而受到抑制。低磷胁迫下根系中酸性磷酸酶活性持续增高,说明其活性与磷供给水平和时间有相关性。[结论]首次克隆鉴定了1个受外生菌根诱导的马尾松紫色酸性磷酸酶基因,其参与了对低磷胁迫的应答,为深刻认识马尾松耐低磷的分子机制、遗传改良提供了新信息和新思路。

Cloning and Expression Pattern Analysis of Ectomycorrhizal Purple Acid Phosphatase Gene in Pinus massoniana Lamb.

[Objective] To study the function of purple acid phosphatase gene and the response of phosphorus deficiency stress in Masson pine (Pinus massoniana). [Methods] The full-length sequence of PAP gene (PmPAP1) was cloned from this germplasm by RACE methodology, subsequently, multiple alignments of amino acid sequences, construction of phylogenetic tree, as well as the bioinformatics analysis were carried out. [Results] The results showed that PmPAP1 was obtained (GenBank accession number: KT390746), whose full-length cDNA sequence was 2,520 bp and the corresponding lengths of open reading frames (ORF) was 1,869 bp, which contained 622 amino acid residues, including a typical conserved domain and belonged to a high molecular weight protein. PmPAP1 had signal peptide and non-transmembrane domain, thus, it was presumably localized in matrix of cytoplasm or organelles, which demonstrated high homology with Nelumbo nucifera Gaertn. and Amborella trichopoda Baill., reflecting the considerably ancient and strict conservation during the evolutionary process. The temporal and spatial expression profiles showed that the expression level of PmPAP1 was induced by ectomycorrhizal symbiosis, which was expressed in different tissues of P. massoniana, the expression level of root was significantly higher than that of stem and leaf. The expression of PmPAP1 was correlated with the phosphorus status of soil in both non-and ectomycorrhizal plants, and its expression might be intensively up-regulated by low phosphorus stress; conversely, high phosphorus level inhibited its expression. Further, the activity of acid phosphatase continued to rise in roots under low phosphorous stress, indicating that the acid phosphatase was time-dependent on the low phosphorus supply. [Conclusion] A PmPAP1 was cloned and characterized for the first time from P. massoniana, especially its enhanced expression pattern by ectomycorrhizal symbiosis under low phosphorus stress, which provided new information about the role of ectomycorrhizal fungi.