Biotechnology Division (CSIR-CIMAP), Central Institute of Medicinal and Aromatic Plants, (CSIR-CIMAP), P.O. CIMAP, Near Kukrail Picnic Spot, Lucknow, 226015, India.
Planta. 2021 Apr 5;253(5):89. doi: 10.1007/s00425-021-03614-3.
BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter. Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3 h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having V of 0.16 µMsec μg protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.
从印度萝芙木中分离得到的 BmG10H-1 转录本具有功能活性。BmG10H-1 启动子驱动 GUS 活性对 MeJA 和创伤的响应。BmMYB35 通过结合其启动子来调节 BmG10H-1 转录本。香叶醇 10-羟化酶（G10H）是重要的调节细胞色素 P450 单加氧酶之一，参与单萜生物碱的生物合成。然而，在印度萝芙木中，G10H 尚未在酶或调控方面得到表征。在本研究中，我们鉴定了两个 BmG10H 的转录本（BmG10H-1 和 BmG10H-2），并对来自印度萝芙木的茉莉酸甲酯（MeJA）和创伤响应的 BmG10H-1 转录本进行了特征描述。在茎中，BmG10H-1 在 MeJA 和创伤处理 3 小时后表现出诱导表达。酵母纯化的重组 BmG10H-1 蛋白具有酶活性，V 为 0.16 µMsec µg 蛋白，并催化香叶醇羟化为 10-羟基香叶醇。通过基因组步行法分离了 BmG10H-1 启动子。BmG10H-1 启动子可在转基因拟南芥中驱动 GUS 表达。与对照未处理的幼苗相比，发现 MeJA 和创伤处理的拟南芥幼苗的 GUS 活性增加，而缺失 MeJA 响应和 W 盒顺式元件的 GUS 活性则没有。这表明 BmG10H-1 启动子含有功能性 MeJA（TGACG）和创伤响应（TGACCT）顺式元件。此外，还纯化了具有茎特异性和 MeJA 响应性的重组 BmMYB35 蛋白，该蛋白与 BmG10H-1 启动子中存在的 MYB 识别顺式元件（TGGTTA）结合，并在酵母中转录激活报告基因。总之，对 MeJA 和创伤响应的 BmG10H-1 的特征描述为其在印度萝芙木中的转录调控提供了新的信息，该调控通过与 MYB 转录因子结合而实现。
