Construction Of CDNA Library Of The Initial Period Of Fruit-body Development For Cordyceps Sinensis And Analysis Of Expressed Sequence Tags (ESTs)

This thesis reviewed the research progress of Cordyceps sinensis in taxonomicstatus, resource distribution, morphology, chemical composition and other aspects upto the present. The necessity to research the critical problem of fruiting bodydevelopment in Cordyceps sinensis was proposal for there were few research on thesexual process. In addition, it also elaborated the EST technology principles, methods,and applications. And the feasibility and rationality of the EST technology wasexplained technically. On the bases above, a clear purpose and significance of thestudy was defined. Finding the key functional genes and revealing the law ofmetabolic regulation in the initiative phase of the fruiting body development inCordyceps fungus by study with molecular biology systematically in order to lay atheoretical foundation for revealling the profound biological mysteries further.The research used the experiment system that fruiting body development ofCordyceps sinensis on artificial medium in the laboratory. The material was a strainseparaed from the Zhayougou, Xiahe County, Gansu Province. It was sampled frombefore the start of the fruiting body development and fruiting body primordia. Thetotal RNA was extracted with the method of Hibind. The mRNA isolated withparamagnetic particle method. The cloning vector cleavaged by enzyme, SMARTcDNA Library Construction Kit used to construct the cDNA library. After selectingthe qualified middle samples and libraries, hundreds of clones were selected to dosequencing. It was made a deep analysis of the sequence measured, including:removing the contaminated sequences to get the high-quality EST sequences. By thesequence clustering method, the same gene EST sequences were clustered together,and then the data of sequence clustering were stitched by sequence in order to get aseries of single genes. On this basis, the single-gene sequences were done local blastgene annotation in the NR and NT database. Those unknown gene were predicted bythe open reading frame (ORF), and the domain structure and function prediction ofthe ORF predicted were done in the interpro database. With the Gibberella zeae as abackground, the three aspects of clustering situation of genes in the biological process,molecular function, and cell component were collected. In addition, it were put intonotes and analysis of the metabolic pathways with the two single gene library ofhighly homologous gene as the KEGG database input. F inally, based on the results ofthe above analysis, the comparison of gene expression were done between the before the start and after the start of the fruiting body development in Cordyceps sinensis.And a deep analysis were made to the different key genes which showed difference instructure and function.The results show that the total RNA obtained in this research have shown goodintegrity, high purity, and the purity of mRNA isolated was therefore qualified. Thereverse transcribed fragments of the cDNA have good integrity. And the further allspecies homology sequence alignment analysis show that the existing sequence hasrelatively high homology with Nectria hacmatococca Berk.et Br., Gibberella zeae andfusarium pseudograminearum. Those single genes which had no sequence homologywere predicted by an open reading frame. It was found6and5strip of encodedproteins from two libraries respectively, most of which contain a significant signalpeptide or a transmembrane structure.The analysis to the two libraries with GO and metabolic network mothed showedthat these genes would have significant function in terms of product molecules,biological processes and biological metabolic pathway. In KEGG, library1and library2were enriched in nucleosome structure function, the electron transport chain andother metabolic pathways.It were found that there were two genes VeA and HSP70which specific expressedwhen the Cordyceps fungus fruiting body development beginning. The RACEtechnique was used to clone cDNA of VeA, length of2515bp, from the total RNA.There greater similarity with other filamentous fungus species in predicted protein. Itproved that Cordyceps sinensis had the gene of VeA.