Investigation of the genes for norlignan biosynthetic enzymes through the large-scale collection of genes expressed in the xylem of Sugi(Cryptomeria Japonica).

在本研究中，我的目的是确定参与的酶在杉（柳杉）降木脂素生物合成的酶基因的分离和酶活性的分析。从杉木的原木上切下一段树干，存放在一个房间里。在收获后第0、10、20和41天收集的边材中的去甲木脂素的分析揭示了从第20天到第41天去甲木脂素、阿加莎树脂酚的显著增加，尽管在第0天未检测到。分别使用来自第0天和第41天边材的cDNA进行抑制消减杂交，并构建了富含在第41天优先表达的基因的cDNA文库。从该文库中，获得了504个非冗余表达序列标签的序列。在这些序列中，有16个编码推测酶的基因与已知序列的相似性被认为与次生代谢有关，其中12个次生代谢相关基因在树干边材中的表达量随着时间的推移而上调。此外，12个基因中的5个在11月的过渡区中表达，此时心材形成被认为正在进行中。考虑到沉香树脂酚是心材成分，推测其中包括参与沉香树脂酚生物合成的酶的基因。利用该重组菌从全长cDNA中制备了酪氨酸3-单加氧酶的重组蛋白，但未检测到酶活性。从植物中分离到酪氨酸3-单加氧酶的报道只有一次，而且是从一种草本植物中分离得到的，为了深入了解该酶在降木脂素生物合成中的作用，以及进一步揭示该酶在木本植物中的作用，有必要对该酶的特性进行研究。

In this research, I aimed to identify enzymes involved in the norlignan biosynthesis in Sugi (Cryptomeria japonica) through the isolation of genes for enzymes and analysis of their enzyme activities. A trunk section was excised from the log of Sugi, and stored in a room. Analysis of norlignans in sapwood collected on Days 0, 10, 20 and 41 post-harvest revealed the prominent increase of a norlignan, agatharesinol, from Day 20 to 41, though it was not detected on Day 0. Suppression subtracted hybridization was performed using the cDNA from the sapwood on Days 0 and 41, respectively, and constructed a cDNA library enriched with genes preferentially expressed on Day 41. From this library, sequences of 504 non-redundant expressed sequences tags were obtained. In these sequences, those for 16 genes encoding putative enzymes were assigned to secondary metabolism based on the similarity to known sequences with predicted functions.The expressions of 12 genes in the secondary metabolism were upregulated in the sapwood of the trunk section with the passage of days. In addition, five of 12 genes were expressed in the transition zone in November when the heartwood formation is assumed to be in progress. Considering that agatharesinol is a heartwood constituent, It was postulated that the genes for enzymes involved in the agatharesinol biosynthesis was included among them. Although the recombination protein of putative tyrosine 3-monooxygenase was prepared from the full-length cDNA using the recombinant bacteria, the enzyme activity could not be detected. There is only one report on isolation of tyrosine 3-monooxygenase from plant, and it was from a herb. Not only to gain an insight into norlignan biosynthesis but also to reveal the role of this enzyme in woody plants, characteristic of the enzyme should be clarified.