Construction Of CDNA Library And Expressed Sequence Tags(EST) Analysis Of Fokienia Hodginsii Leaf

Fokienia hodginsii (Dunn) Henry et Thomas, parker of cupressaceae Fokienia hodginsii, is a national secondary protection plant, also is unique precious tree species in China. Based on overview of fokienia hodginsii resources, biological characteristics and ecological habits, ecological landscape application value, breeding and cultivation techniques to summarize, in a deeper molecular cloning, metabolic pathway and the study of protein function is basically blank, remains to be further explored.The research adopted several methods with total RNA extraction, and establish a cDNA library and the analysis of EST function. The main conclusions as follows:1、The experiment extracted total RNA of Fokienia hodginsii with five methods of CTAB, Trizol, CTAB-LiC1,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 CTAB-LiCl that can reach the experiment requirement. However, it 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 Fokienia hodginsii.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. The optimal system was built with 2μL ds cDNA, the 18th cycles of LD-PCR and the ratio between vector and insert (cDNA) that was 1 to 1.96. The titer of the library was 3.6×106cfu·mL-1,the percent of the recombination rate is 84.6. The size of the inserts in the library varied from 0.4-2kb. The titer of the amplified cDNA library was 5.61×107cfu·m L-1.3^ There were 212 ESTs that were obtained. The percent of the content of GC was 46.8, and the averge length was 530.83bp all in the 212 ESTs.77 unigenes were generated after clustering, The redundancy of the library was the percent of 53.3. There were 29 low expression abundance genes,41 moderate expression abundance genes and 7 high expression abundance genes, respectively accounted for 37.66%,63.25%,9.09% in the unigenes.4、Through the 77unigenes homology analysis and sequence alignment, 10unigenes had not significant homology,13% is the new sequence; Article 67 significant homology, proportion is 87%, which contains the proteins of known functions were 14, accounted for 18.2%, and predict protein article 38, accounted for 49.4%, and the unknown protein, article 15 (19.4%).5、compared with the database of Blast2GO, gain a comprehensive understanding of the gene function annotion.77 unigenes in blast2GO get number of comments GO term for a total of 162, the average each sequence to get 3.136 GO terms. And summed up in three categories:Biological processes, Molecular Function, cell components.6、In species similarity analysis aspect, the leaf of Fokienia hodginsii and Picea sitchensis has the highest similarity, but did not detect the similarity with the same genus, this plant in molecular biology research is less.