Detection And Identification Of Microrna In Apple And Prediction Of Precursor Structure

miRNAs are small, non-coding regulatory RNAs which come from endogenous eukaryotic genome and play important regulatory roles in many biological processes as posttranscriptional regulators of gene expression. miRNA which also play an important role in plant growth and development, control development of plants, flowering timing, metabolism, stress response and so on. Approach of miRNA regulate gene is a major biological discovery in the field, to demonstrate a new mode of gene regulation. In this paper, species and their conservation of miRNAs, and the effects to miRNA expression between in vitro plants and in vivo plants were completely studied by using'Hanfu'apple as materials. According to EST database information and bioinformatics software, related miRNA precursor structure were predicted in apple. The miRNAs with expressed difference were found and their probable functions in apple growth and development were analyzed. The main results were as follows:1.154 miRNAs were checked in apple plants by gene chip. There were 67 miRNAs whose intensity value of fluorescent signals maintained from 400 to 1500 and 87 miRNAs beyond 1500. Among them, the 100 miRNAs from miRBase represented 35 miRNAs gene families. miR159 represented the largest miRNA family in M. domestica, which had twelve members. There were eleven members for miR171 family in M. domestica. miR166 had eight members. miR164 had seven members. miR167 and miR169 all had six members. miR160 and miR319 all had five members. miR156, miR168, miR172, miR398 and miR399 all had three members. miR157, miR393 and miR478 all had two members. Other 19 miRNA families including miR161, miR162, miR165, miR390, miR391, miR394, miR395, miR396, miR397, miR407, miR414, miR416, miR426, miR447, miR477, miR479, miR529, miR530 and miR535, only had one member.2. According to the miRBase Registry, the 35 miRNA families checked in apple were compared with other nine plants, such as Arabidopsis, rice, populus and so on. Twenty-five familes (miR156, miR157, miR159, miR160, miR162, miR164, miR165, miR166, miR167, miR168, miR169, miR171, miR172, miR319, miR390, miR391, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR414 and miR447) were also detected in A. thaliana, and ninteen families of them (the exceptions were miR157, miR165, miR168, miR391, miR414 and miR447) were completely conserved in rice. Two rice miRNA families (miR416 and miR426) were also present in apple. And 23(miR156, miR157, miR159, miR160, miR162, miR164, miR166, miR167, miR168, miR169, miR171, miR172, miR319, miR390, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR477 and miR478) of these familes were also detected in P. trichocarpa.3. Fourteen kinds of conserved miRNA sequences were identified by stem-loop RT-PCR in leaves of'Hanfu'apple. The expression of five conserved miRNAs between leaf, tender stem, young fruit and root of apple was studied by stem-loop RT-PCR. The expression of six conserved miRNAs between shoot, one-year branch, two-year branch and perennial branch of apple were studied by stem-loop RT-PCR. While the technology system with stem-loop RT primers was the suitable method for Qualitative study of miRNAs in apple plants.4. Semi-quantitative RT-PCR with stem-loop RT primers was used to investigate the expression difference of ten miRNAs between in vitro plants and in vivo plants of'Hanfu' apple. The obvious expression difference was found in apple plants with two propagation mammers, miR156 and miR157 were obviously upregulated in the young leaves of in vitro plants.5. According to EST database information and bioinformatics software, eleven related miRNA precursor structure were predicted in apple, and the eleven putative miRNA could be classified as being representatives of nine different miRNA families. Precursor structure shows they have the characteristic fold-back structure.