Analysis Of MicroRNA Expression During The Embryonic Development Of Chinese Horseshoe Crab

Chinese horseshoe crab(Tachypleus tridentatus)is the rare and endangered aquatic arthropods.The process of it`s embryonic development has been made clear but the mechanism of molecular regulation is unclear.This study is to explore the crucial miRNAs during embryonic development of Chinese horseshoe crab and reveal its developmental regulatory mechanism.The results of this study wll be useful for reference in its resources recovery and providing important information for deep research of miRNAs.In this study,parental Chinese horseshoe crabs were collected from Fujian coastal waters and reared in tank indoor.Natural ecological habit and environment was simulated to induce Chinese horseshoe crabs pairing and laying eggs.Four different developmental stages of embryos were adopted to research which were the first embryonic molting(Tt01),the second embryonic molting(Tt02),the third embryonic molting(WT03)and the fourth embryonic molting(WT04).The Tt01,Tt02 and WT03 are belong to the organ specialization.The WT04 is belong to the rapid growing stage.The depth sequencing of the RNA materials for these four embryonic developmental stages was conducted through Solexa technology.The sequencing results were statistically analyzed,then the sequencing fragments were screened by Rfam,and repeat sequence were deleted,finally,all the fragments were classified,and all miRNAs including known and unknown miRN were found in four development periods.The expression profiling of them is built,then differential expression profiling was built.Differential expression analysis and conserved analysis were conducted on known miRNA,and unknown miRNAs were predictive analyzed including secondary structure prediction of their precursors,differential expression analysis and there target gene prediction and functions prediction.The results are summarized as follows:1.Identification and difference expressions of the known miRNAThe known miRNA expression profile is built.There are 9 known mi RNAs in all o f four stages.The four mi RNAs including bmo-miR-10-5p_L+1?bmo-miR-10-3p_L-1R-1?lmi-mi R-9a-5p_R-1and tca-miR-9a-5p_R+1 are higher but difference in the four stages.T he miR-10 family is related with embryo angiogenesis and tissue growth.The miR-9a fa mily is related with embryo tissue differentiation.2.Predicting the New miRNA4835 new miRNAs were discovered from the four development stages in this study.198 miRNA sequence were conserved in the phylum Arthropoda,but the pre-mi RNA sequence had different nucleotide consisting in different species.37 miRNA in the 198 unknown miRNA were selected as candidates for analysis.The 37 miRNA were high abundance and the miRNA expression profiling was built.Part of miRNA were estimated to relate with Chinese horseshoe crabs embryos differentiation and development,which named miR-375,miR-184,miR-7,let-7,miR-92 a,miR-2a,miR-133 and miR-34.miR-375 was related with tissue growth;miR-184 and miR-7 were related with tissue differentiation;let-7 was related with late embryo develops into trilobite larvae;miR-92 a was related with angiogenesis;miR-2a was related with the apoptosis of embryonic cells;miR-133 was related with skeletal muscle cell differentiation in embryonic cells;miR-34 was related with the growth and spread of cancer cells.There were 89 new mature miRNA sequences which can't be mapped to the current published Arthropoda genomes,these miRNAs were not be analyzed in the paper.There were4548 mature sequences and pre-miRNA sequences which were not reported.Among them,15 miRNAs in abundant expression were selected as candidates and expression profiles were constructed.Secondary structures were predicted by using RNAfold software.The results showed that these 15 miRNAs had two stem-loop structures.The target genes of 13 mi RNAs has been predicted.3.Verify some of miRNAsFour differentially expressed miRNA in Tt01 and Tt02 stages were selected to analyze by real-time quantitative RT-PCR technique to get the differentially expressed multiple in Tt01,Tt02 stages,and compared with solexa sequencing results.The results showed that solexa sequencing results were quite reliable.