【作者】 王菁；

【导师】 陈新；

【作者基本信息】 成都中医药大学 ， 生药学， 2009， 硕士

【摘要】 银杏又名白果、公孙树、.鸭脚子、鸭掌树,是新生代第四纪冰川期的孑遗植物,为我国独存的珍稀名贵树种。银杏叶提取物(GBE)具有促进血液循环,改善心血管功能,清除体内超氧离子,预防肿瘤等作用,特别是在治疗和预防心脑血管系统疾病方面具有重要的作用,银杏的开发和研究已经成为整个人类共同努力的课题。银杏制剂的质量主要取决于银杏叶的质量,不同产区的银杏叶功能活性成分含量差距较大,说明药用银杏品种的选择与银杏制剂的质量有着直接联系。通过先进的基因工程技术有目的性的培育高产银杏药用品种已经成为开发银杏资源的一种重要途径。而进行基因改造的前提是要充分了解有效成分的代谢途径中的关键酶基因和起主要调控物质的基因,而克隆关键酶基因是首要任务之一。本研究通过分子生物学技术对银杏黄酮类活性成分代谢途径中的第一个关键限速酶苯丙氨酸解氨酶(Phenylalanine ammonia-lyase, PAL)进行了较为系统的分子生物学研究：对银杏基因组进行酶切、连接接头、特异PCR扩增,运用基因步移技术,对银杏PAL基因侧翼序列进行了克隆,获得长度为588 bp的基因片段,通过在线比对鉴定为PAL基因的一部分,所得序列已登录在NCBI的GenBank中,核酸序列Accession number为EU713843。利用先进的生物信息学分析方法和丰富的银杏EST数据资源,初步建立了较完整和系统的利用EST资源挖掘新基因的方法,通过对EST序列的比对、筛选、祛除冗余、拼接和聚类得到了2条银杏PAL基因家族新成员的电子克隆片段。并设计了特异PCR引物,通过PCR扩增验证了电子克隆片段的真实存在性。证实了2条银杏PAL新成员真实存在于银杏基因组中,并为进一步开展银杏PAL基因家族研究奠定了一定的实验基础。更多还原

【Abstract】 Ginkgo biloba L., also known as ginkgo, maidenhair tree, duck foot trees, is a relict plant of the Cenozoic Quaternary ice age, for our own survival of rare and valuable species. Ginkgo biloba extract (GBE) can promote blood circulation, improve cardiovascular function, remove the body of superoxide ions and prevent tumor, especially it has an important role of the treatment and prevention of cardiovascular and cerebrovascular diseases. The exploitation and study of Ginkgo biloba has become the subject of the joint efforts of the whole humanity.The quality of ginkgo preparations depends on the quality of the ginkgo leaves.The content of the functional activated components of ginkgo in different area is very different, the choice of varieties of medicinal ginkgo has a direct connection with The quality of ginkgo preparations.Cultivating high-yielding varieties of medicinal ginkgo purposely has become an important way of the exploitation of ginkgo through advanced genetic engineering techniques. The precondition of gene modification is to understand the key enzymes and major regulation and control genes which be in active components’s metabolic pathway, and the cloning of key enzymes gene is the first thing.A systematic study of molecular biology of the first key rate-limiting enzymes phenylalanine ammonia-lyase (PAL) in the active components of ginkgo flavonoids metabolic pathway was carried out through molecular biology technology. Using digestion, joints, specific PCR amplification, gene walking techniques, Ginkgo PAL gene flanking sequence was cloned. A length of 588 bp gene fragment was obtained which was identified a part of PAL gene through on-line BLAST. It was enrolled in the GenBank of NCBI, the accession number is EU713843.By the advanced bioinformatics analysis method and the rich resources of ginkgo’s EST data, A more complete and systematic method of discovering new gene in the EST gene resources was established. Two gingko new PAL gene family members in silico cloning fragments were obtained by EST sequences matching, screening, eliminating redundancy, splicing and polying. The PCR amplification verified the two new members of PAL were existed in genomes of Ginkgo biloba by designed specific PCR primers, It is the base of the further study about ginkgo PAL gene family.更多还原