【作者】 杨成君；

【导师】 王军； 穆立蔷；

【作者基本信息】 东北林业大学 ， 森林植物资源学， 2008， 博士

【摘要】 人参为五加科多年生草本植物,是我国常用的珍贵药材。人参种质资源主要包括野生人参和栽培人参,野生人参为国家珍稀濒危物种。近年来,栽培人参品质下降、品种混乱、参地连作障害、生产滞销等原因严重破坏了人参种质资源。急需构建人参种质基因库,收集和整理人参宝贵的种质资源。人参作为珍贵的中药材资源,其体内次生代谢产物（人参皂苷等）具有多种生理功能,具有重要的药用价值。人参功能基因和次生代谢生物合成途径的研究相对较少,完全的基因组和草图序列还有待于建立。因此,保存人参种质资源和加速基因水平的研究进程具有重要意义。本文进行了人参叶片cDNA文库的构建,在此基础上进行了EST的初步分析,部分基因的时空表达分析,利用现有的人参EST资源进行SSR信息分析,人参EST-SSR标记的建立等方面的研究,结果如下:通过四种RNA提取方法提取人参叶片总RNA,其中SDS法能分离出高质量的RNA,可以进行下一步的分子生物学研究;以四年生红果人参叶片为材料,提取叶片总RNA合成cDNA,连接到质粒载体pDNR-LIB上。采用电穿孔法将重组质粒转化到DH5a中,成功构建了人参叶片cDNA文库。经文库质量鉴定表明:原始文库滴度为1.008×10⁶ pfu·mL^-1,扩增后的文库滴度为2.968×10⁹ pfu·mL^-1,重组率接近100%,插入片段大小范围在0.5～2 kb之间,平均约为0.85 kb,表明应用SMART技术已成功构建了红果人参叶片cDNA文库,可供进一步的EST测序分析使用。对四年生红果人参叶片cDNA文库测序获得441条有效EST序列。GenBank登录号:ES554557-ES554997。441条EST拼接后代表了354个Unigenes。BLAST注释结果表明具有已知或推测功能基因136个,未知功能基因127个,未比对上的基因81个（可能是新基因）。根据BLASTX注释与GO分类结果,并参照MIPs分类标准对354个Unigenes按照生物学功能划分为9个大类:能量与代谢（energy/metabolism,13.8%）、蛋白合成（proteinsynthesis,5.9%）、信号传导（signal transduction,4.8%）、细胞救援与防御（cellrescue。defence,3.1%）、运输（transport,4.8%）、光合作用（photosynthesis,1.9%）、转录（transcription,2.2%）、蛋白活性调节（protein activity regulation,1.69%）、其他（others,61.5%）。采用荧光定量RT-PCR技术研究人参叶、根部的部分基因的表达方式。结果表明,人参中的同一基因在叶、根部的表达模式不完全相同。所选择的基因的表达分别在人参皂苷生物合成途径、三羧酸循环途径、信号传导、抗逆、苯丙氨酸代谢途径等植物次生代谢中发挥重要作用。对人参基因时空表达的研究,将有利于我们进一步研究它们在人参代谢活动过程中的基因表达调控机理。利用现有的7055条人参EST序列搜索出791个SSR,其出现频率为11.21%,平均长度为21.37 bp,平均分布频率为1/5.7 kb。人参EST-SSR的重复类型比较丰富,一至六核苷酸都能观测到。二核苷酸重复是主要的重复类型,占全部EST-SSR的56.89%,其次是三核苷酸重复的占全部SSR的21.11%。AT、GAA是二核苷酸和三核苷酸中出现次数最多的重复基元类型,分别占28.89%和10.18%。表明人参EST中的SSR数量非常丰富。本研究对现有人参EST资源中的SSR进行了分析,明确人参EST中SSR发生频率和分布特征信息,了解人参EST资源的特性,从而为建立其EST-SSR标记及其在人参分子标记的理论研究、人参遗传育种中的应用提供重要的依据。根据搜索出的SSR设计了68对EST-SSR引物,对测试样品进行PCR扩增并检测多态性。分别以人参品种集安长脖和抚松二马牙的DNA为模板,对设计的EST-SSR引物进行筛选,发现有43对EST-SSR引物能扩增出产物。在9个人参品种、2个西洋参品种和2个刺五加品种中进一步对这些可扩增的引物对进行多态性检测,发现有26对引物显示多态性,占可扩增引物的60.47%,占设计引物总数的38.23%。表明实验建立的EST-SSR标记是一种行而有效的方法,是对现有人参EST资源的进一步开发和利用。建立这种标记对于加速人参EST资源的开发利用、丰富其分子标记类型与绘制遗传图谱、实现特定性状的辅助选择和遗传多样性等研究都具有重要的意义。目前人参中还没有建立EST-SSR标记的报道,本研究根据现有人参EST资源首次建立了EST-SSR标记。更多还原

【Abstract】 Ginseng（Panax ginseng C.A.Meyer）,a perennial herb from the Araliaceae family,is one of the most commonly utilized medicinal plants.Ginseng germplasm resources mostly involve in wild ginseng and cultural ginseng.The wild ginseng is considered as rare endangered species in China.At present,Ginseng germplasm resources are destroyed because of quality dropped, variety confusion,injury by continuous cropping,produce dull sale.ect.So we need to construct the gene library of ginseng germplasm,collecte and rearrange the precious germplasm resources.Ginseng as rare traditional Chinese medicinal materials,their secondary metabolite（ginsengoside etc.） in vivo are important effective constituents in biological activity of ginseng and possess important medicinal value.The research about functional gene and secondary metabolism biosynthetic pathway is relatively less in ginseng.In case of Panax ginseng,Hawerever,the full genome and draft sequences are yet to be established.It is meaningful for preserving panax ginseng germplasm resources and accelerating to studying the gene level process.In this paper,we reported the construction of the cDNA library from panax ginseng leaves,EST analysis,space-time expression analysis of gene,analysis of SSR of EST resources in panax ginseng,development of EST-SSR marker in panax ginseng.The main results are as follows.RNA was isolated from panax ginseng leaves using four methods of extraction（CTAB, Trizol,Bizol,SDS）.Highly-grade RNA was isolate by using SDS method,better than the other methods and suitable to the demands of molecular biological research.The four years old red fruit panax ginseng was used as experimental material,and the total RNA was extract from the leaves,the cDNA was synthesized and the ds cDNA fragment was ligated into the pDNR-LIB vector.The recombinant plasmid was transformed into E.coli DH5 a by electroporation.The library qualification evaluation showed:the titer of primary cDNA library was 1.008×106 pfu·mL^-1,the titer of amplified library was 2.968×109 pfu·mL^-1,the percentage of recombination was about 100%,the fragment size of inserted was 0.5～2kb.The average insert size was 0.85 kb.The cDNA library was constructed successfully,and could be satisfied for further studying on expressed sequence tags（ESTs） analysis.A total of 441 high quality ESTs were obtained from cDNA library of panax ginseng leaves.The GenBank accession numbers are ES554557-ES554997.The 441 ESTs were assembled into 354 unigenes.The results of BLAST annotation showed:36 known or putative functional gene,127 unknown gene,81 no hits（maybe new genes）.The 354 unigenes were divided into 9 functional categorier based on BLASTX,GO analysis and MIPs Functional Catalogue,i.e.,energy/metabolism（13.8%）, protein synthesis（5.9%）,signal transduction（4.8%）,cell rescue,defence（3.1%）, transport（4.8%）,photosynthesis（1.9%）,transcription（2.2%）,protein activity regulation （1.69%）,others（61.5%0）.To studying gene expressed mode between roots and leaves of panax ginseng in different growth time,eight genes from panax ginseng were selected for expression analysis using real time revers transxription-polymerase chain reaction（RT-PCR）.The results showed the expressed mode of the same gene was different or incomplete the same.Eight genes studied played the important roles in ginsenoside biosynthetic pathway,tricarboxylic cycle,signal conduct,stress-tolerance,phenylalanine metabolic pathway and so on.To study gene spacetime expressed in panax ginseng,there will be benefit to further study regulation and control mechanism of gene expression in metabolic activity.Seven hundred and ninety one microsatellites（SSRs） were isolated from 7055 panax ginseng expressed sequence tags（ESTs）.The occurrence frequency of SSR was 11.21%,the average length searched was 21.37 bp and the average distance of distribution was 1/5.7 bp. The dinucleotide repeat was the dominant repeat type in panax ginseng EST-SSR,accounting for 56.89%of the total EST-SSR,and then trinucleotide repeat accounts for 21.11%of the total EST-SSR.AT and GAA were the most frequent motifs,accounting for 28.89%and 10.18%（?） dinucleotide and trinucleotide repeats,respectively.SSR quantities were abundant in panax ginseng ESTs,SSR occurrence frequency and distributional characteristixs of EST resources were definited,which will provide important references for development of EST-SSR marker and application in inherit breeding of panax ginseng.According to primer design criteria,sixty-eight primer pairs for EST-SSR were designed. Under an appropriate PCR reaction system,all EST-SSR primer pairs were screened ag（?）inst genomic DNA of ji’anchangbo and fusong’ermaya from panax ginseng respectively,and fortythree of sixty-eight EST-SSR primer pairs resulted in PCR products.Then all forty-three EST-SSR primer pairs available were detected in nine Panax ginseng cultivars,two Panax quinquefolius cultivars and two Acanthopanax senticosus cultivars for polymorphisms,and twenty-six EST-SSR primer pairs were polymorphic,accounting for 60.47%of primer p（?）s amplified,accounting for 38.23%of total primer pairs designed.These results showed that it is an effective and feasible approach to develop EST-SSR markers using Panax ginseng ESTs. Developing of ginseng EST-SSR markers will further detect and make full use of ginseng ESTs resources.It is meaningful for accelerating to exploiting and utilizing of the ginseng ESTs resource,enriching molecular marker types,constructing genetic map,assessing genetic diversity,and assisting plant breeding.At present,there is no report about developing of ginseng EST-SSR marker in domestic and foreign.更多还原