巴西橡膠樹一個新的法尼基焦磷酸合酶基因的克隆及表達分析

郭冬等

摘 要 利用同源克隆的方法在橡膠樹中獲得一個新的法尼基焦磷酸合酶基因，命名為HbFPS2。序列分析顯示，HbFPS2基因組序列長4 171 bp，由12個外顯子和11個內含子組成，cDNA全長1 288 bp，包含1 029 bp的開放閱讀框、105 bp的5′-UTR和154 bp的3′-UTR，編碼342個氨基酸，推測分子量為39.6 ku，等電點為5.27。氨基酸序列比對顯示，HbFPS2具有FPS家族保守的5個結構域。進化分析結果表明，HbFPS2和HbFPS1親緣關系最近，與大戟科的蓖麻、大戟、麻風樹法尼基焦磷酸合酶聚為一個亞類。定量PCR結果表明，HbFPS2在橡膠樹根、樹皮、葉、花和膠乳等組織中均有表達，在花和膠乳中表達量較高，在根中表達量最低；茉莉酸和乙烯處理均能提高HbFPS2基因的表達量，處理9 h時基因表達量分別提高了36倍和8倍。結果為分析法尼基焦磷酸合酶基因表達特性及其在天然橡膠生物合成途徑中的作用機制奠定基礎。

關鍵詞 巴西橡膠樹；法尼基焦磷酸合酶；特異表達；茉莉酸；乙烯

中圖分類號 S794.1 文獻標識碼 A

Abstract Farnesyl pyrophosphate synthase is a key enzyme in nature rubber biosynthesis in Hevea brasiliensis. In this study， a new gene coding for farnesyl pyrophosphate synthase， designated as HbFPS2， was isolated by homology-based candidate gene method in Hevea brasiliensis. Sequence analysis revealed that the HbFPS2 genome sequence is 4 171 bp， contained 12 exons and 11 introns. The full-length cDNA of HbFPS2 is 1 288 bp with 1 029 bp open reading frame， encoding 342 amino acid residues with molecular mass of 39.6 ku， and pI 5.27. Multiple sequence alignment showed that the HbFPS2 included 5 domains with highly conserved amino acids. The phylogenetic analyses revealed that HbFPS2 belong to plant farnesyl pyrophosphate synthase family， in which Hevea brasiliensis， Ricinus communis， Euphorbia pekinensis and Jatropha curcas were clustered into one clade. The results of real time PCR analysis indicated that HbFPS2 expressed at different levels with the highest transcription in the flowers， followed by the latex， and lowest in the roots. The transcription of HbFPS2 was induced in the latex by jasmonic acid and ethylene， increased 36 and 8-fold respectively. This research lays a foundation for studying the gene expression pattern and regulating mechanisms of HbFPS2 in nature rubber biosynthesis.

Key word Hevea brasiliensis； Farnesyl pyrophosphate synthase； Differential expression； Jasmonic acid； Ethylene

doi 10.3969/j.issn.1000-2561.2015.03.001

橡膠樹法尼基焦磷酸合酶（Farnesyl pyrophosphate synthase，FPS）是天然橡膠生物合成途徑中的關鍵酶之一。巴西橡膠樹（Hevea brasiliensis）是天然橡膠的主要來源。天然橡膠是異戊二烯代謝途徑的一個重要產物之一，同位示蹤術和核磁共振成像技術研究結果表明，法尼基焦磷酸（Farnesyl pyrophosphate，FPP）是橡膠生物合成的起始物[1]。法尼基焦磷酸合酶（Farnesyl pyrophosphate synthase，FPS，EC2. 5. 1/EC2. 5. 1. 10）是植物類異戊二烯合成途徑中的一個重要關鍵酶，催化兩分子的異戊烯基焦磷酸和一分子的二甲基丙烯基焦磷酸以1，4頭尾連續縮合反應，形成15碳的FPP[2]。FPP還是植物體內甾醇、三萜、長醇、泛醌、類胡蘿卜素等許多萜類衍生物質合成的前體物和蛋白質法尼基化的底物[3]。……