橡胶树果糖-1,6-双磷酸酯酶基因的克隆及表达特性分析

龙翔宇1, 曹冰2, 梁启福2, 方永军1, 戚继艳1, 唐朝荣1,*
1中国热带农业科学院橡胶研究所, 海南儋州571737; 2海南大学农学院, 海口570228

通信作者:唐朝荣;E-mail: chaorongtang@126.com;Tel: 0898-23307223

摘 要:

果糖-1,6-双磷酸酯酶(FBPase)是糖异生途径中的关键酶, 在糖代谢中起重要作用。本研究首次从橡胶树中克隆并获得胞质型HbFBPase基因, 其全长cDNA序列1 541 bp, 包含一个1 017 bp的开放阅读框, 编码一个由338个氨基酸残基组成的蛋白质。氨基酸同源性分析发现, 该蛋白含有保守的FBPase活性位点、腺嘌呤核糖核苷酸(AMP)结合位点及金属离子结合位点。生物信息学分析显示, HbFBPase蛋白无导肽酶切位点, 不具有跨膜结构域, 且为亲水蛋白, 推测该蛋白可能在细胞质中发挥作用。实时荧光定量PCR分析表明, HbFBPase基因在叶中高表达, 胶乳及种子次之。进一步分析HbFBPase基因在非光合库组织胶乳(产胶细胞的细胞质)中的表达模式, 结果显示该基因表达受割胶、伤害处理调控, 推测其在橡胶树胶乳糖代谢中发挥重要的调控作用, 参与了橡胶烃的生物合成调控。此外, HbFBPase基因表达受多种植物激素如乙烯(ET)、脱落酸(ABA)、茉莉酸(JA)、植物生长素(2,4-D)、水杨酸(SA)及赤霉素(GA)的调控。本研究初步揭示HbFBPase是橡胶树胶乳糖异生作用的关键酶, 是胶乳糖酵解及橡胶合成的负调控因子, 为探讨胶乳糖异生与橡胶烃合成之间的关系提供理论基础, 有助于进一步了解胶乳糖代谢的调控机理。

关键词:橡胶树; HbFBPase; 基因克隆; 生物信息学分析; 基因表达

收稿:2013-07-02   修定:2013-10-21

资助:“863”计划课题(2013AA102605)、国家自然科学基金(31170630)、海南省自然科学基金(312029)和中国热带农业科学院橡胶研究所基本科研业务费(1630022011022)。

Molecular Cloning and Expression Analysis of a Fructose-1,6-Bisphosphatase Gene from Rubber Tree (Hevea brasiliensis Muell. Arg.)

LONG Xiang-Yu1, CAO Bing2, LIANG Qi-Fu2, FANG Yong-Jun1, QI Ji-Yan1, TANG Chao-Rong1,*
1Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China; 2College of Agronomy, Hainan University, Haikou 570228, China

Corresponding author: TANG Chao-Rong; E-mail: chaorongtang@126.com; Tel: 0898-23307223

Abstract:

Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of the gluconeogenic pathway and involved in glucose metabolism. The cDNA sequence of a cytosolic HbFBPase was isolated and analyzed in Hevea brasiliensis for the first time. HbFBPase cDNA contained a 1 017-bp-long open reading frame (ORF) that encoded a protein of 338 amino acids. Bioinformatics analyses were performed on HbFBPase protein. Hb-FBPase had highly conserved cytosolic FBPase active site, adenosine monophosphate (AMP) binding site and metal binding site. Due to the characteristics of high hydrophilicity, no signal peptide and transmembrane domains, it was inferred that HbFBPase functioned mainly in the cytoplasm. Real-time PCR analysis revealed that HbFBPase highly expressed in leaves, latex and seed. HbFBPase expressions were affected in the latex by tapping and wounding, which suggests that HbFBPase plays a role in the regulation of sugar metabolism, especially in rubber biosynthesis. In addition, the expression of HbFBPase was induced by some hormones, such as ethylene (ET), abscisic acid (ABA), jasmonic acid (JA), auxin (2,4-D), salicylic acid (SA) and gibberellin (GA). Briefly, this study has provided clues about the relationship between gluconeogenic and rubber biosynthesis in latex, and will be helpful to further understand the mechanism of latex glucose metabolism.

Key words: Hevea brasiliensis; HbFBPase; gene cloning; bioinformatics analysis; gene expression

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