【作者】 刘文哲；

【导师】 安利佳；

【作者基本信息】 大连理工大学 ， 生物化工， 2002， 博士

【摘要】 本论文的研究目的是利用基因工程手段培育转基因植物，使其降低木质素含量、提高纤维素含量，从而解决造纸制浆过程中因降解木质素造成的污染问题，提高原材料利用率。 本论文运用了一种以PCR为基础的快速克隆植物全长cDNA的方法。该方法利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法可直接获得目的基因全长序列，避免了复杂的构建、筛选cDNA文库的过程。本论文利用此方法首次从紫穗槐中克隆了与纤维素合成前体----尿苷二磷酸葡萄糖的代谢有关的尿苷二磷酸葡萄糖焦磷酸化酶(UGPase)基因和与木质素合成相关的4-香豆酸：CoA连接酶(4CL)基因的全长cDNA序列，并由此推测出相应的氨基酸序列。 所获得的紫穗槐尿苷二磷酸葡萄糖焦磷酸化酶基因UGPA长1832bp，其中开放阅读框1416bp，编码471个氨基酸，与其它植物的UGPase基因的序列相比有较高的同源性。推测的氨基酸序列含有保守的Lys残基(Lys258，Lys324，Lys362，Lys404，Lys405)，是典型的UGPase蛋白。 所获得的4-香豆酸：CoA连接酶(4CL)基因长1911bp，开放阅读框1623bp，编码540个氨基酸。序列相似性检索结果表明，紫穗槐4CL全长cDNA与其它植物的4CL基因的序列有较高的同源性。推测的氨基酸序列含有预计的AMP-binding位点PYSSGTTGLPKG、催化活性反应区GEICIRG和保守的Cys残基，是典型的4CL蛋白。 以植物表达载体pCAMBIA1301为基础，构建了UGPA基因的植物表达载体pCAUGPA，还构建了4CL基因反义植物表达载体pCA4CL。用农杆菌介导法将这两种基因转入烟草和杨树，经抗生素筛选和分子检测得到了转基因植株。同时，还用木醋酸菌来源的UGPase基因转化烟草，获得了转基因植株。 检测转4CL反义基因烟草的木质素含量，发现木质素含量降低39.6％。研究了木醋酸菌来源的UGPase基因与植物来源的UGPA基因在转基因烟草中的表达，测定了两种转基因烟草的尿苷二磷酸葡萄糖焦磷酸化酶的活性、木质素含量和纤维素含量。结果表明，两种转基因烟草的尿苷二磷酸葡萄糖焦磷酸化酶的活性均有明显提高；转紫穗槐UGPA基因的烟草纤维素含量有所提高，木质素含量略有降低，纤维素与木质素含量之大连理工大学博士学位论文比提高;而转木醋酸菌来源的UGPase基因的烟草木质素含量和纤维素含量均显著提高。更多还原

【Abstract】 The two most abundant components of the wood fiber are cellulose and lignin. While the cellulose is the valuable component for the pulp and paper industry, removal of the lignin is the most energy intensive and environmentally damaging step of the pulping process. Ideally we would like to identify and propagate a tree that produces a maximal amount of wood with a reduced lignin content and increased cellulose content by biotechnology.A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGPase), a key enzyme producing UDP-glucose in the synthesis of sucrose and cellulose, was cloned by using this method. Another Amorpha fruticosa cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned ,too.A full-length UGPase cDNA was obtained. The size of the cDNA fragment is 1832 bp, which included an 1416 bp open reading frame and encoded a peptide of 471 amino acids. The deduced amino acid sequence exhibits significant homology with the UGP genes cloned from other plants.A full-length 4CL cDNA was obtained. The size of the cDNA fragment is 1911 bp, which included an 1623 bp open reading frame and encoded a peptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins.The sense expression plasmid of UGPA and the antisense expression plasmid of 4CL gene were constructed with the target gene trans-inserted into plant expression vector pCAMBIA1301 respectively. Transgenic tobaccoand poplar plants were obtained by regeneration of agrobacterium-mediated transformed leaves. PCR and Southern blot analyses confirmed the integration of target gene in transformed tobacco genome. We evaluated lignin content severely altered in the expression of transgenic tobacco with pCA4CL antisense constructs and pCAUGPA constructs. The Klason lignin level of anti-4CL-transformed tobacco was much lower than that of the control. The cellulose content increased in transgenic tobacco with UGPA constructs.We also obtain transgenic tobacco transformed with Acetobacter xylinum UGPase gene. Compared the lignin and cellulose level of these two kinds of transgenic tobacco, we found that the cellulose content increased and the lignin content deduced in transgenic tobacco transformed with Amorpha fruticosa UGPA constructs, both the cellulose content and the lignin content increased in transgenic tobacco transformed with Acetobacter xylinum UGPA constructs.更多还原