Production mechanism of β-thujaplicin by cell cultures of woody species

β-木柏苷（β-Thujaplicin），日语称为扁柏醇（hinokitiol），是植物中的一种tropolone，具有广泛的抗真菌和抗菌谱，有助于许多柏科物种的心材经久耐用。本文研究了柏科植物卢西塔尼柏（Cupressus lusitanica）细胞培养产生β-thujaplicin的机制。首先，研究了从细胞表面的激发子受体到β-thujaplicin生物合成开始的信号转导途径。加速剂和/或抑制剂喂养实验表明，g蛋白、脂氧合酶和蛋白激酶参与了信号转导，cAMP和Ca离子作为信使。jasmonate通路被认为在导致β-thujaplicin产生的转导系统中起重要作用。萜类油烯合成酶被发现是最有可能参与中间单萜烯生物合成的酶，而单萜烯最有可能是β-胡贾林辛的直接前体。利用RT-PCR和cDNA筛选对该酶基因进行克隆，我们发现该酶在激发子的作用下被激活，因此我们从经过激发子处理的芦西塔菌细胞的cDNA文库中获得了3个可能编码萜类油烯合成酶的DNA克隆。然后，通过微阵列方法搜索参与β-thujaplicin生物合成的基因s，因为期望通过诱导诱导的mRNA(s)反映β-thujaplicin生物合成基因(s)。在lusitanica的cDNA文库中，有48个克隆在激发子处理前后表现出显著的强度差异。这些克隆的功能有待在今后的工作中进一步研究。

β-Thujaplicin, called as hinokitiol in Japanese, is one of plant tropolones, contributing to the durability of heartwood of many Cupressaceae species due to its broad antifungal and antibacterial spectra. We have studied the mechanism of the production of β-thujaplicin by cell cultures of Cupressus lusitanica, a species in the Curpressaceae family.At first, a signal transduction pathway from elicitor receptor(s) on the cell surface to the onset of β-thujaplicin biosynthesis was investigated. It was elucidated by accelerator and/or inhibitor feeding experiments that G-protein, lipoxygenase, and protein kinases are involved in the signal transduction, and cAMP and Ca cation functioned as messengers. The jasmonate pathway is suggested to play an important role in the transduction system leading to β-thujaplicin production.Terpinolene synthase was found to be the most probable enzyme that is involved in biosynthesis of an intermediate monoterpene, most probably the direct precursor of β-thujaplicin. For cloning the gene of this enzyme by means of RT-PCR and cDNA screening, we obtained three DNA clones that may code the terpinolene synthase from a cDNA library of an elicitor-treated cell culture of C.lusitanica, because it was found that the enzyme was activated by the action of the elicitor. Then, the gene(s) that is involved in the β-thujaplicin biosynthesis was searched by the micro array method, for it was expected that mRNA(s) induce by the elicitation should reflect the β-thujaplicin biosynthesis gene(s). In cDNA library of C.lusitanica tells, it was observed that 48 clones exhibited significant differences in the strength between before and after the elicitor treatment. Function of these clones should be studied in future works.