杨树漆酶基因调控木质素聚合和次生壁形成机制

漆酶是木质素单体氧化聚合成木质素过程中的一个关键的酶类。本项目以杨树为材料，在前期工作的基础上，首先通过使用荧光定量PCR、GFP荧光定位等技术，分析3个漆酶LAC4、LAC16和LAC29基因在木质部发育各阶段的组织特异性表达与细胞器定位规律；然后通过转基因技术在杨树体内过量或缺失表达这3个漆酶基因，通过拉曼光谱、核磁共振、透射电镜、转录组等技术分析杨树木质部木质素含量和单体组成的变化，探讨该基因过量或缺失表达对杨树木质素生物合成和木质部次生壁形成的影响机制；同时利用抗体柱技术，从植物体内纯化分离获得漆酶蛋白，同时使用悬滴法对纯化等到的3个漆酶蛋白进行结晶，并通过X-射线衍射法对晶体结构进行解析，在体外酶蛋白水平上研究酶对不同底物的生化特性与功能；从而全面阐述杨树漆酶基因参与杨树木质素聚合、木质部次生壁加厚与木材形成过程中的表达与调控机制，为林木木材形成与品质控制提供理论基础。

Laccase is a large gene family which is the key enzymes in lignin oxidation polymerization in plant. To understand the mechanism for laccase in lignin synthesis in poplar, the tissue specific expression of three lactase, LAC4, LAC16 and LAC29, during the development of xylem of poplar would be analyzed using fluorescence quantitative PCR and GFP fluorescence localization. To determine the function of three laccases during lignin oxidation polymerization in xylem, the expression of lactase should be changed in P. tomentosa using an antisense or.overexpression strategy, respectively. The content of lignin and S/G ratio in xylem of transgenic populous should were analyzed using Raman spectroscopy and Nuclear magnetic resonance (NMR) and xylem differentiation and the secondary wall development should were also visualized by Transmission electron microscopy (SEM). Meanwhile, the enzyme activity would be analysis after purity using antibody column technique. At the same time, three purified laccase proteins were crystallized by suspension drop method, and the crystal structure was analyzed by X-ray diffraction. At last, the role of laccase participated in xylem differentiation and lignin synthesis would also be analysis by RNA-seq technology. It would be used to more understand the molecular mechanisms of laccase in poplar lignin polymerization, xylem secondary wall thickening and wood forming.