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

在这项研究中，我旨在通过分离酶基因并分析它们的酶活性来鉴定Sugi(Cryptomeria Japan Onica)中与去甲木脂素生物合成相关的酶。从杉木的原木上切下一段树干，存放在一个房间里。对采后0天、10天、20天和41天采集的边材中去甲木脂素的分析表明，从第20天到41天，去甲木脂素显著增加，但在第0天没有检测到。分别在第0天和第41天用边材中的基因进行抑制消减杂交，构建了一个富含第41天优势表达基因的cDNA文库。从该文库中获得了504个非冗余表达序列标签序列。在这些序列中，根据与已知的具有预测功能的序列的相似性，将16个编码可能酶的基因归类为次生代谢，其中12个次生代谢基因的表达随着时间的推移在树干边材中上调。此外，12个基因中的5个在11月份的过渡区表达，当时心材的形成被认为正在进行中。考虑到刺梨脂醇是一种心材成分，推测参与刺梨脂醇生物合成的酶的基因也在其中。利用重组菌获得了酪氨酸3-单加氧酶的全长重组蛋白，但未检测到该重组蛋白的酶活性。目前从植物中分离得到的酪氨酸3-单加氧酶只有一种，而且是从一种中草药中分离得到的。为了深入了解去甲木脂素的生物合成，也为了揭示该酶在木本植物中的作用，必须弄清该酶的特性。

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.