【作者】 蔡文伟；

【导师】 张树珍；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2006， 硕士

【摘要】 龙血树是热带、亚热带地区所特有的珍稀植物,是著名民间药物“血竭”的重要原料。该药在我国中药材市场中占有重要的地位。目前血竭的生产是从多年生的龙血树上取其带脂茎杆提炼而成,该法需要大量的原材料,不断的砍伐已使其资源面临枯竭的威胁,现其已被列为国家三级保护的濒危植物。如何有效地开发和利用龙血树这一宝贵的资源,使其得到可持续和高效的利用是目前研究的一个重要方向。通过分离和鉴定龙血树血竭生物合成相关的基因将有助于揭示龙血树血竭形成的机理,进而为提高龙血树血竭的含量和发明新的血竭生产方式提供理论基础。本研究以经诱导物诱导处理刚产生血竭和未经诱导物处理的海南龙血树(Dracaena cambodiana Pierre ex Gagnep)组培苗为材料,利用抑制消减杂交(SSH)技术构建了龙血树血竭生物合成相关基因的差减cDNA文库,采用PCR方法筛选差减文库获得431个重组子。随机挑取200个克隆经Reverse Northern Blot筛选剔除假阳性后,共获得59个差异较明显的阳性克隆,测序后得51条差异片段,通过BLASTn和BLASTx进行比较分析,结果表明:分离到与信号转导相关的cDNA片段5个,转录翻译相关蛋白cDNA片段5个,物质能量代谢酶类cDNA片段12个,另外有23个克隆在Genbank中均未发现显著相关的同源序列,推测它们可能功能未知的新基因或是靠近3’-末端的基因片段,因保守性较低,难以通过同源比较推测其功能。更多还原

【Abstract】 Dracaena draco Linn. is the peculiarly rare plant of tropical and subtropical area. It is the important material of famous civilian medicament dragon’s blood. The dragon’s blood plays an important role in the Chinese medicinal materials market. At present, the dragon’s blood is extracted from the stem with lipid of perennial Dracaena draco Linn., and the method needs many raw materials. The Dracaena draco Linn. is faced with dried up menace because of continuing lopping, and it has been to the endangered plant under third grade state protection. How to effective exploit and utilize the resource of Dracaena draco Linn., and to be continuable and high effective using are the important aspect of research. To isolate and identify the genes related to dragon’s blood biosynthesis from Dracaena draco Linn. will help to reveal the mechanism of dragon’s blood forming,and to offer theory base for increasing the content of dragon’s blood and inventing the new dragon’s blood production mode.The study is based on the materials which are induced by inducer and non-induced from the Dracaena cambodiana seedlings in tissue culture. The study is initiated by construction a subtraction cDNA library of genes related to dragon’s blood biosynthesis from Dracaena draco Linn. by using suppressive subtractive hybridization (SSH) methodology, then 431 recombinants are got by screening the subtraction cDNA library with PCR. To randomly pick the 200 clones by Reverse Northern Blot, and to eliminate the pseudo positive, then 59 positive clones of obvious difference are got, while after sequencing, 51 different fragments are got. To comparatively analyze the different fragments by BLASTn and BLASTx, the result shows that: 5 cDNA fragments related to signal transduction, 5 cDNA fragments related to transcription and translation protein, 12 cDNA fragments related to substance and energy metabolism enzyme, and other 23 cDNA fragments which can not be found from the Genbank with the significantly related homologous sequence. To speculate that they may be the new genes of unknown function or gene fragments nearing 3’-end, and the homology is very low, so it is difficult to speculate the function by homology comparison.更多还原