The Isolation Of MiRNAs From Chinese Mitten Crab(Eriocheir Sinensis) And Analysis Of Their Expession During The Oocyte Maturation

microRNA(miRNA) is a kind of small endogenous RNA, which is22~24nt in length.Mature miRNAs come from pre-miRNA cut by Dicer and form the RISC,target with the3’UTR of mRNA and inhibit their expression or translation.Recently,there are a largenumber of miRNAs cloned in the ovary of various species.However,most of the ovarianmiRNAs are not distinct in the function.So far,there are the largest number of miRNAsfound in the insecta,43miRNAs found in the daphnia of crustacean,while no research arereported in the shrimp and crab.Eriocheir sinensis play an important role in the aquatic economy.So far,there are aseries of research focusing on the histology level and cytology level.To study the role ofmiRNAs playing during the process of oocyte maturation,we obtain the small RNA lowerthan36nt by Solexa sequencing from four development stage(previtellogenesis,vitellogenesis, germinal vesicle and germinal vesicle breakdown),and blast with the sangermiRbase database.We use the genome of daphnia and drosophila as a reference,predictingthe miRNA candidate on the basis of forming the stem-loop structure. We obtain220miRNAs,62known miRNAs belonging44family and136miRNA candidates.The lengthdistribute two peak,the21nt to the24nt and the25nt to the26nt,meaning that there maybegenerous piRNAs.In the62known miRNA,the abundance of miR-184is highest and theabundance of miR-100and let-7are lower,meaning that these miRNAs may executeimportant function during the oocyte maturation.Most of small RNA molecules are21~24nt and25~26nt in length,maybe there are piRNA molecules. Blasted with the sequence and conservation of mature miRNA,62known miRNAsare classified into44families. The result show that19miRNA families are exist in thevertebrate and invertebrate,23miRNA families and two miRNAs belong with no familyare only exist in the invertebrate.During the23families and2miRNAs,only7families andmiR-2779are only in the arthropods,meaning that these miRNAs may be important for theArthropods,such as molting.While miR-252family and miR-71family are cloned merelyin the Branchiostoma and invertebrate.We presumed that the two families eliminatethrough selection or competition.Most of the miRNAs are conserved in the vertebrate andinvertebrate,such as miR-1and let-7.bantam and miR-2279have a mutation in the seedregion,while miR-315and miR-8*have mutation in other reigion.In addition,we choose the mRNA sequences which total length is greater than1000bpand3’UTR greater than300bp for target prediction.The function of these mRNA containthe maturation of ovary,the development of gonads,transcriptional control,immunizationand so on.Cyclin B and cdc2kinase are the component of MPF of the crab ovarymaturation.There are three box elements cloned in the3’UTR of cyclin B mRNA,which areconfirmed to mediate down-regulation.As the miRNAs are conserved among thespecies,the regulation maybe also conserved.Accordingly,we presume that the cyclin Bmaybe the target of miRNAs. We obtain the three miRNAs binding with three kinds ofbox,which are miR-2,miR-7and miR-79.The numbers of miRNA target with cyclin B are19,containing54bingding sites.MiR-2target with K-box have higher MFE,which is-24.0kcal/mol,and miR-2*is-24.5kcal/mol.The MFE of miR-7target with GY-box is-21.7kcal/mol,and the MFE of miR-79target with Brd-box is-17.0kcal/mol.The numbers ofmiRNA target with cdc2kinase are eight,containing ten binding sites.miR-252a have thelowest MFE,-27.2kcal/mol.We use qRT-PCR to detect the expression of three miRNAs during oogenesis,evenoocyte maturation.The result of real-time PCR show that miR-2are highly expressed inpVt,then have a servere descent in the Vt and a slow rise in the GV.Finally the miR-2havethe highest level in GVBD.The expression of miR-7has no significance change,while an apparent upgrade in the GVBD.The expression of miR-79has no significancechange,while an apparent discent in the GVBD.According that cyclin B have the sameexpression profile,the miR-2may regulate the cyclin B expression in the protein level. ThemiR-7have an upgrade in GVBD,and may regulate cyclin B protein expression.WhilemiR-79have a discent in GVBD and need a additional experiment to validate its functionon cyclin B expression.miR-79have a significante change in GVBD and maybe affectoocyte maturation through other path.In the three miRNAs,the MFE of miR-2target with cyclin B is-24.0kcal/mol,andmiR-2*may have a coordinated regulation. Therefore the miR-2is choosen to be theoptimum selection.Meanwhile,we cloned3’UTR of cyclin B including three boxes in to the3’UTR of luciferase of pgl-3vector.After sequencing,we found that there only twoK-boxes.We contransfect the UTR vector and miRNA-2mimics into the293Tcell,discovering that the activity of luciferase apparently descend21.19%,meaning thatcyclin B may be the target of miR-2.But how the miR-2regulate the cyclin B expressionstill need an additional verification in protein level.