Construction Of Panax Ginseng CDNA Library, EST And Relative Gene Expression Analysis And Development Of EST-SSR Markers

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.