Tasnim Sifat, Gries Regine, Mattsson Jim
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.
Plants (Basel). 2020 Aug 12;9(8):1018. doi: 10.3390/plants9081018.
Upon harvest, Western redcedar (WRC; ) trees have a high incidence and extent of heartwood rot. While monoterpenoids and lignans have been linked to rot resistance in this species, other specialized metabolites, such as diterpenes, are likely to contribute to rot resistance. Here we report the cloning and functional assessment of three putative diterpene synthase (TpdiTPS) genes expressed during heartwood formation in WRC. The predicted proteins of the three genes lack either of the two catalytically independent active sites typical of most diTPS, indicating monofunctional rather than bifunctional activity. To identify potential catalytic activities of these proteins, we expressed them in genetically engineered strains that produce four potential substrates, geranylgeranyl diphosphate (GGDP), and normal stereoisomers of copalyl diphosphate (CDP). We found that TpdiTPS3 used GGDP to produce CDP. TpdiTPS2 used normal CDP to produce levopimaradiene. TpdiTPS1 showed stereoselectivity as it used normal CDP to produce sandaracopimaradiene and -CDP to produce -stemod-13(17)-ene. These genes and protein enzymatic activities have not been previously reported in WRC and provide an opportunity to assess their potential roles in heartwood rot resistance in this economically important species.
收获时，西部红雪松（WRC）树木的心材腐朽发生率高且范围广。虽然单萜类化合物和木脂素与该物种的抗腐性有关，但其他特殊代谢产物，如二萜类化合物，可能也有助于抗腐。在此，我们报告了在西部红雪松心材形成过程中表达的三个假定二萜合酶（TpdiTPS）基因的克隆和功能评估。这三个基因的预测蛋白缺乏大多数二萜合酶典型的两个催化独立活性位点中的任何一个，表明其具有单功能而非双功能活性。为了鉴定这些蛋白的潜在催化活性，我们将它们在基因工程菌株中表达，这些菌株可产生四种潜在底物，香叶基香叶基二磷酸（GGDP）、柯巴基二磷酸（CDP）及其正常立体异构体。我们发现TpdiTPS3利用GGDP生成CDP。TpdiTPS2利用正常的CDP生成左旋海松二烯。TpdiTPS1表现出立体选择性，因为它利用正常的CDP生成山达海松二烯，利用 -CDP生成 -异海松-13(17)-烯。这些基因和蛋白质的酶活性此前在西部红雪松中尚未见报道，为评估它们在这个经济重要物种的心材抗腐性中的潜在作用提供了机会。
