树木形成层及木质部细胞骨架与木材形成机理及基因调控

4-coumarte: coenzyme A ligase is a key enzyme of general phenylpropanoid metabolism which.provides the precursors for a large variety of important plant secondary products. The mRNA and.genomic sequences encoding 4-coumarte:coenzyme A ligase ( 4CL1 ) is cloned from Populus tomentosa..There are four introns in the 4CL1 DNA sequences.The reconstructed 4CL1 protein were one-step.purified from denatured inclusion-body heterologusly overexpressed in Escherichia coli by his-tag.affinity chromatograpy. The reconstructed 4CL1 protein exhibited highly substrate preference. Based on.国家自然科学基金资助项目结题报告.5.the cloned and sequenced cDNA from Populus tomentosa, we have obtained a deducted amino acid.sequence of 4CL1 (the primary structure of 4CL1) and folded its the secondary and advanced 3D.structure in varies pattern with the computer softwares. The relationship between structure and function.of the enzyme for substrates has been optimalized and approached. It has been theoretically proven that.4-coumarte is the natural substrate of the 4CL1 based on its higher fitness and affinities of enzyme.structures to the substrate..Intermediate vector was constructed by inserting 4CL1 gene into vector pBI121 to produce a.non-tissue-specific constitutively active promoter (CaMV35S-4CL1) driving vector. Regenerated.transgenic tobacco plants obtained by transformation were detected by PCR and southern bloting to.verify and select the putative transgenic plants. The activity of enzyme and the content of lignin and.cellulose in transgenic plants were analyzed. Intermediate vectors were constructed by inserting 4CL1.promoter fused with the 4CL1 gene into vector pBI101 and GRP1.8 promoter fused with 4CL1 gene and.into vector pBI101 to produce tissue-specific expressed promoter-driving vector (4CL1promoter-4CL1,.GRP1.8-4CL1). Transgenic tobaccos were obtained The activity of enzyme and the content of lignin and.cellulose were analyzed. We found that the activity of 4CL1 enzyme was increased 30-40%.

该项目研究了杨树形成层区细胞的活动规律，克隆获得形成层特异表达的启动子和基因，构.建正义、反义融合表达载体转化植物，在维管组织区域定位定位表达启动子来驱动与植物（树木）.生长和木材形成关键组分（纤维素、木质素等）生物合成关键酶，调控形成层区细胞的分裂和分.化，实现外源基因在植物（树木）生长与木材品质形成中的组织定位表达，达到定向调控树木生.长和木材品质定向改良的目的，终实现树木生长和木材品质定向改良的目标。.4-香豆素辅酶A-连接酶是木质素合成途径中的调控酶，决定木质素合成中最后一步的反应。.研究中发现，在调节该酶的含量能够有效的调节木质素的含量。使用能特异性形成层定位表达的.启动子在转基因植物形成层特异性定位表达4-香豆素辅酶A-连接酶可以有效调控木质素的生成。.用生物信息学方法对4CL1 蛋白三维空间结构进行了模拟，成功得到该蛋白的理论三维空间结.构模型。同时模拟了该蛋白三维结构与底物相互作用的过程，发现该蛋白对4－香豆酸结合匹配程.度最高，该结构与该酶对4－香豆酸催化活性最高的结果一致，从而初步解释了该酶的催化机理。