Construction Of CDNA Library And Expressed Sequence Tags(EST) Analysis Of Sarcandra Glabra Leaf

Sarcandra glabra (Thunb.) Nakai, an evergreen subshurb belonging to the genus Sarcandra of Chloranthaceae, is a traditional Chinese Herbal Medicine. The paper has summarizd S. glabra from six aspects, including Research on Materia Medica, Biological Characteristics, Resources Overview, Chemical Component, Pharmacologic Action and Research Status. Current research is focused on Cultivation Techiques, Chemical Component, Quality Control and Genetic Diversity of S. glabra. However, the research is nearly blank on molecular clone, metabolic pathways and protein function in the deeper level, which needs to be further explored.The research constructed a full-length-cDNA library,and then made the preliminary bioinformatics analysis on base of an appropriate total RNA extraction of S. glabra. The main results were as follows:1、The experiment extracted total RNA of S. glabra with six methods of CTAB, CTAB-Trizol, Trizol, CTAB-LiCl,TIANGEN RNAprep Pure Plant Kit and Bio Teke RNApure TotalRNA Plant Kit.The result showed that it was a good way to extract total RNA with TIANGEN RNAprep Pure Plant Kit that can match the experiment requirement and cost higher. Meantime, the CTAB-LiCl method, which spent too much time and had a relatively complicated process, was also a choose that has a lower cost.The other methods were unsuited to extract total RNA of S. glabra.2、A full-length-cDNA library was constructed with the required total RNA. In the process of synthesis of cDNA, it was better to optimize initial materials of ds cDNA, the optimal LD-PCR cycles and the ratio between vector and insert.2μL ds cDNA and the 18th cycles of LD-PCR were the optimal factors and the ratio between vector and insert (cDNA) that was 1 to 1.98. The titer of the library was 1.14×107 cfu·mL-1. The size of the inserts in the library varied from 0.5-3 kb, and the average of the inserts were about 1000bp. The titer of the amplified cDNA library was 0.94×108 cfu·m L-1.3、There were 177 ESTs that were obtained. The percent of the content of GC was 41.9, and the averge length was 790bp all in the 177 ESTs.151 unigenes were generated after clustering, including 12 contigs and 139 singletons. The redundancy of the library was the percent of 14.7.There were 139 low expression abundance genes,10 moderate expression abundance genes and 2 high expression abundance genes, respectively accounted for 92.05%,6.62%,1.32% all in the unigenes.4、Through the homologous analysis of the 151 unigenes,32 unigenes had no homology, which were new sequences; 119 unigenes were homologous, covering 24 known functional proteins,91 predicted proteins and 4 unknown proteins.5、In order to gain a comprehensive understanding of the gene function annotion and metabolic pathways, it needs to use the two softwares of Blast2GO and KEGG.119 unigenes with known and putative function were distributed into functional categories with Blast2GO and generated 415 GO terms, which were divided into 3 categories. Biological Process was the major functional category, containing 185 GO terms(44.85%). Second larger functional category was Cellular Component, constituted 126 GO terms(30.36%).104 GO terms(25.06%) were classfied into the Molecular Function category.There were 50 sequences with annotation in the database of KEGG. Next, it should classify these sequences in accordance with BRITE function level. It had gross 126 sequences that had been counted up. Metabolism was the major functional category, containing 29 sequences; Second larger functional category was Genetic Information Processing, constituted 19 sequences; 10 sequences were classfied into the Environmental Information Processing; 11 sequences were concluded Cellular Processes category; 25 sequences were related to Organismal Systems; The last category was about Human Diseases,32 sequences were detected.6、In the aspect of species distribution about similarity by GO annotation, the leaf of S. glabra was similar to Nelumbo nucifera. But it did not detect the other plants in the same genus which showed that few research had been studied on genome,transcriptome and proteome.