Cloning And Identification Of Nocoding Small Rnas In Eisenia Fetida

MicroRNAs (miRNAs) are approximately 20-25 nucleotides (nt) noncoding RNAs that have found in various eukaryotes. They down-regulate posttranscriptional of gene. Mature miRNAs are processed by Dicer from precursors (pre-miRNAs) which have 70-90 basepair and hairpin secondary structure. They have the capability of affecting translation of target mRNAs by entirety or not binding to complementary sequences at the 3' untranslated region, which induce degradation or translational repression. As a sort of important regulatory molecule, they paticipate in multifarious of regulatory pathways, including cell growth and differentiation, cell proliferation and apoptosis, fat metabolism, tumorigenesis and so on. Recently researches show that scientists have found other short noncoding RNAs (Piwi-interacting RNA, piRNA), which strikingly different from miRNAs in their length. They are about～30 nt, and originally found in germ cells of mammal. These small RNAs act regulatory function by combining with PIWI proteins which belong to Argonaute protein family. They play important roles in mRNA stabilization, protein synthesis, chromatin framework and structure of genome. Their idiographic function and biogenesis are in research, because of studying of piRNAs are in primary phase.At present, the study of small noncoding RNAs are developing rapidly. MiRNA database has 9539 entries miRNA, including from lower organisms as virus, bacteria and algae to higher organisms as human, chimpanzee and so on. But piRNA database only has six species involving human, mouse, rat, drosophila, zebrafish and platypus. Scientists have found miRNAs in Capitella sp. (polychaete annelid), but without piRNAs. Earthworm is oligochaete annelid and hermaphrodite. It possesses strongly regeneration ability, so we choose Eisenia fetida as reseach object. Owing to the genome of Eisenia fetida has not sequencing, identification of miRNAs and piRNAs are difficult, as well as very few reports. Earthworm is one of important regeneration pattern organisms. It has important significance for studying miRNA and regeneration associated miRNA. In this study, we construct a cDNA library of earthworm's small RNA, and clone portion of miRNAs and piRNAs. This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in regeneration.At first, total RNA was isolated from sexual maturation animals using RNAiso reagent as described in the main text. Then, 16-30nt small RNA was purifyed on 10% denaturing polyacrylamide, 8 M urea gel. Third, 3' adapter and 5' adapter ligation, and purify after each ligation. Forth, RT-PCR and PCR products purify. Fifth, the PCR products were digested, concatemerized and tailed A. At last, was cloning into T vectors, and construct a cDNA library by transformating competent cells of TSS method.The colonies were first screened by blue/white spot, then PCR screening of bacterial clonies eliminate fake clonies. We choose 43 colonies for sequencing, and abtain 20 single sequences. Sequences were subjected to BLAST search for E. fetida miRNA homologs against EST and nr databases, miRNA and piRNA databases, then using Mfold 3.2 or RNA Strucrue to predict secondary structures, and analyze A+U content. Finally, 8 candidate miRNAs, 7 candidate piRNAs and 5 small nocoding RNA were obtained.Analyze miRNA expression using real-time quantitative PCR and the 2^-ΔΔCt Method in different tissue. Using 5.8S rRNA as endogenous control, efe-let-7, P2B7, P2B10 and P2B1-1 as target gene. The result shows that expression levels of regeneration tissue are more than normal tissue, and each of small RNA is different, expression level of P2B10 is higher, while expression level of P2B1-1 is lower.