Zhang Mengyan, Wang Siyao, Yin Jing, Li Chunxiao, Zhan Yaguang, Xiao Jialei, Liang Tian, Li Xin
College of Life Science, Northeast Forestry University, Harbin, 150040, China.
State Key Laboratory of Tree Genetic Breeding, Northeast Forestry University, Harbin, 150040, China.
Protoplasma. 2016 Sep;253(5):1347-63. doi: 10.1007/s00709-015-0893-3. Epub 2015 Oct 10.
Betula platyphylla is a rich repository of pharmacologically active secondary metabolites known as birch triterpenoids (TBP). Here, we cloned the squalene synthase (SS) and squalene epoxidase genetic (SE) sequences from B. platyphylla that encode the key enzymes that are involved in triterpenoid biosynthesis and analyzed the conserved domains and phylogenetics of their corresponding proteins. The full-length sequence of BpSS is 1588 bp with a poly-A tail, which contained an open reading frame (ORF) of 1241 bp that encoded a protein of 413 amino acids. Additionally, the BpSE full-length sequence of 2040 bp with a poly-A tail was also obtained, which contained an ORF of 1581 bp encoding a protein of 526 amino acids. Their organ-specific expression patterns in 4-week-old tissue culture seedlings of B. platyphylla were detected by real-time PCR and showed that they were all highly expressed in leaves, as compared to stem and root tissues. Additionaly, both BpSS and BpSE were enhanced following stimulation with ethephon and MeJA. The expression of BpSS was enhanced by ABA, whereas BpSE was not. The SA treatment did not affect the BpSS and BpSE transcripts notably. Using a genome walking approach, promoter sequences of 965 and 1193 bp, respectively, for BpSS and BpSE were isolated, and they revealed several key cis-regulatory elements known to be involved in the response to phytohormone and abiotic plant stress. We also found that the BpSS protein is localized in the cytoplasm. Opening reading frames of BpSS and BpSE were ligated into yeast expression plasmid pYES2 under control of GAL1 promoter and introduced into the yeast INVScl1 strain. The transformants were cultured for 12 h, the squalene content of galactose-induced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the triterpenoids biosynthesis of B. platyphylla. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the biosynthesis of birch triterpenoids.
白桦是一种富含药理活性次生代谢产物的植物，这些次生代谢产物被称为白桦三萜（TBP）。在此，我们从白桦中克隆了鲨烯合酶（SS）和鲨烯环氧酶基因（SE）序列，它们编码参与三萜生物合成的关键酶，并分析了其相应蛋白质的保守结构域和系统发育。BpSS的全长序列为1588 bp，带有一个poly - A尾巴，其中包含一个1241 bp的开放阅读框（ORF），编码一个由413个氨基酸组成的蛋白质。此外，还获得了BpSE的全长序列，为2040 bp，带有一个poly - A尾巴，其中包含一个1581 bp的ORF，编码一个由526个氨基酸组成的蛋白质。通过实时PCR检测了它们在4周龄白桦组织培养幼苗中的器官特异性表达模式，结果表明与茎和根组织相比，它们在叶片中均高度表达。此外，用乙烯利和茉莉酸甲酯刺激后，BpSS和BpSE均增强。ABA增强了BpSS的表达，而BpSE则未增强。水杨酸处理对BpSS和BpSE转录本没有显著影响。采用基因组步移法分别分离了BpSS和BpSE的965 bp和1193 bp的启动子序列，它们揭示了几个已知参与植物激素应答和非生物胁迫的关键顺式作用元件。我们还发现BpSS蛋白定位于细胞质中。将BpSS和BpSE的开放阅读框连接到GAL1启动子控制下的酵母表达质粒pYES2中，并导入酵母INVScl1菌株。将转化体培养12小时，通过高效液相色谱（HPLC）检测方法，半乳糖诱导BpSS表达的酵母细胞中鲨烯含量是对照（空载体对照酵母细胞）的13.2倍。并且，诱导BpSE表达的酵母细胞的鲨烯环氧酶活性约为对照的11.8倍。这些结果表明我们克隆的白桦BpSS和BpSE确实参与了三萜类化合物的合成。这是首次报道克隆白桦的SS和SE，对于理解SS和SE在白桦三萜生物合成中的调控作用可能具有重要意义。这是首次报道克隆白桦的SS和SE，对于理解SS和SE在白桦三萜生物合成中的调控作用可能具有重要意义。
