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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla
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Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla

机译：白桦角鲨烯合酶和角鲨烯环氧酶基因的分子克隆和启动子分析

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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.
机译：白桦（Betula platyphylla）是富含药理活性次生代谢产物的仓库，称为桦木三萜（TBP）。在这里，我们从白僵菌中克隆了角鲨烯合酶（SS）和角鲨烯环氧化酶遗传（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，带有一条poly-A尾巴，其中包含一个1241 bp的开放阅读框（ORF），编码413个氨基酸。此外，还获得了带有poly-A尾部的2040 bp的BpSE全长序列，该序列含有1581 bp的ORF，编码526个氨基酸。通过实时荧光定量PCR检测了在白桦芽胞杆菌4周龄组织培养幼苗中它们的器官特异性表达模式，表明与茎和根组织相比，它们均在叶片中高表达。另外，用乙烯利和MeJA刺激后，BpSS和BpSE均得到增强。 ABA增强了BpSS的表达，而BpSE则没有。 SA处理并未显着影响BpSS和BpSE转录本。使用基因组步移方法，分离出BpSS和BpSE的启动子序列分别为965和1193 bp，它们揭示了几个关键的顺式调控元件，这些元件已知参与了对植物激素和非生物植物胁迫的反应。我们还发现BpSS蛋白位于细胞质中。在GAL1启动子的控制下，将BpSS和BpSE的开放阅读框连接到酵母表达质粒pYES2中，并引入到酵母INVScl1菌株中。将转化体培养12小时，通过高效液相色谱（HPLC）测试方法，半乳糖诱导的BpSS表达酵母细胞中鲨烯的角鲨烯含量是对照（空载体对照酵母细胞）的13.2倍。并且，诱导的BpSE表达酵母细胞的角鲨烯环氧酶活性是对照的约11.8倍。这些表明我们克隆了确实参与三萜类化合物合成的桦树BpSS和BpSE。这是第一份克隆了白桦的SS和SE的报道，对于了解SS和SE在白桦的三萜类生物合成中的调控作用可能具有重大意义。这是第一份克隆了白桦的SS和SE的报道，可能对于了解SS和SE在桦木三萜类生物合成中的调控作用具有重大意义。

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