Cloning And Primary Defination Of The Free Nuclear Division Stage Ovule MiRNA In Pinus Tabulaeformis Carr

MiRNA is a class of endogenous noncoding small RNA with about 22nt length, not only specific in organ, but also expressive in organ specific developing period, As a new regulate small RNA, miRNA play an important role in the regulation of growth and development of organisms.In order to study the regulatory effects of miRNA in the development of Pinus tabulaeformis Carr. ovule, the Pinus tabulaeformis Carr. ovule in April at free nuclear division stage was selected as the study materials.Used direct cloning method and bioinformatics techniques in this research, the results were as follows:1.SDS mehtod, CTAB method, Trizol method and improved SDS method were employed to isolate the total RNA. The quality of RNA was examined with agarose gel electrophoresis and the OD with ND-2000 spectrophotometer. The results indicate that the purity of RNA extracted by improved SDS method was significantly satisfactory for the demands molecular biological research. A260/A280 was 1.974, A260/A230 was 2.082. the sharpness of 28S bands was twice than 18S of bands.2. By optimizing and verifing the method of enrichment,isolation and recovery of small RNA, the cDNA library of small RNA was constructed, and the small RNA cloned technology of Pinus tabulaeformis Carr. ovule was well developed through the process of researching.3. By disposing the sequence by DNAman and blasting to EST database from NCBI,8 small RNA sequences were obtained which could complementarity to the database, finally 2 candidate miRNA sequences were selected after predicting second structure pre-miRNA by Mfold,named miRNA-c1,miRNA-c2.