Analysis On The Growth Characteristics And Regulatory Mechanisms In Macrobrachium Nipponense Based On The Pedigree Construction,Transcriptome And Proteome

Macrobrachium nipponense is a kind of fresh water prawn of Palaemonidae with important economic values.For the very low natural yield and the extreme difficulty in artificial breeding,it is difficult to obtain both healthy and fast-growing larvae in aquaculture with high quality.No large-scale production to meet the needs of market demand is a limiting factor for farming Macrobrachium nipponense.Otherwise,the traditional way to get larvae by catching wild berried females for their natural reproduction in the earth ponds is an inefficient way and gained low quality larvae.We did this study for the cultivation of standardization and got high quality larvae,further improve the production of the oriental river prawn and further study of the molecular mechanism of growth regulation.In this study,we focus on the growth characteristics of the oriental river prawn and build its full-sib families as the experimental material.The influence of environmental factors in the growth process must be eliminated.We obtained the main regulation networks of growth rate cotrolled in Macrobrachium nipponense and scanned the growth-related newly mi RNAs by transcriptome and mi RNAome in nucleic acid level and proteomic mass spectrometry analysis from the protein levels then combination analysis with the three.We also find the differences between the hepatopancreas and muscle by paraffin sectioning in the histology level.It is as a reference for the further breeding of high-yield and fast-growing new strains of Macrobrachium nipponense.The main contents and results of this study are as follows:?1?With the use of artificial bionic aquatic plants,this research achieved the whole development process of larvae,artificial breeding of Macrobrachium nipponense and establishment of the pedigree in the aquarium.It is provid a reference to standardized construction of pedigree of Macrobrachium nipponense indoor for selecting breeding.?2?At the same time,an unnamed new species in the family Palaemonidae was found and carried on the preliminary study to its establishment of the pedigree.It will be the new parents in the future to improve the breeding of Macrobrachium nipponense for its good growth characteristics.The following experiment was carried out successively and get the members of full-sib family cultured in the same aquarium at their 70 days after birth in the molt interphase as experimental materials.The selected members were divided into two groups with significantly different body lengths.FG group shows the fast-growth and slow-growth for SG group.?3?Histological study:With the adult male and female Macrobrachium nipponense as the reference,the structure of cross section of hepatopancreas was compared between FG and SG group by paraffin section.The 4 types of cells in hepatopancreas of FG group have more complete differentiation than SG group.The connective tissue and embryonic cells in SG group is more than FG group,it reflects the differences efficiency of digestion and nutritional intake between the two groups.It reveals tha the metabolism of FG group is more vigorous.Paraffin section results of abdominal muscles transverse section showed that the two parameters of the abdomen muscle fibers in FG group longer than SG group and showed that the abdominal muscles grow faster in FG group.?4?Transcriptome and mi RNAome research:Total RNA were extracted from the integer larvae.Based on the High-seq 4000 platform transcriptome sequencing and the High-seq 2500 platform to small RNAs sequencings,the differentially expressed of genes and mi RNAs were screened,GO enriched and KEGG pathway analysis.The target genes of the screened mi RNA were predicted.The correlation between differentially expressed genes and mi RNAs was analyzed either.The q RT-PCR method was taken for verifing the differentially expressed levers between DEG and the DEM.?1?No reference genome transcriptome sequencing:There were 22792166 clean reads were get from FG group,97.33% in the total reads;22534835 clean reads were got from SG group,97.32% in the total.2739 significantly differentially expressed genes are both in FG group and SG group.SG group as the reference group,it has 2116 genes significantly up-regulation expression in FG group and there are 623 genes expression is significantly down-regulation.Selected 15 differentially expressed genes to verify the high-throughput sequencing results by q RT-PCR.The q RT-PCR results are basically consistent with the transcriptome sequencing,very strong correlation between them?R2 = 0.901?.?2?mi RNAs sequencing :The analysis results show that the DEM were 592 include new mi RNA,differentially expressed in both two groups.457 mi RNAs are expressed common in both groups.There are68 of mi RNAs in the FG group only,67 mi RNA only expressed in the SG group.84 new mi RNAs differentially expressed were scaned.There are 15 new mi RNAs expressed in the FG group only,18 new mi RNAs only expressed in SG group.In order to verify the accuracy of the sequencing results,the expression of 11 elected mi RNAs for the q RT-PCR verified and the result is basically identical with the high-throughput sequencing and the correlation coefficient is R2 = 0.972.?3?The correlation analysis between the transcriptome and small RNA sequencing:14 major differences expressed mi RNAs?5 up and 9 down expressed include the two new screened mi RNAs named novel₂2 and novel₅1?.Correlation analysis results show that the 5 up mi RNAs control 45 genes down expression and 9 down expressed mi RNAs control137 genes up expression.?5?The mass spectrum analysis of Proteomic:For verifying the expression after gene transcription,the total proteolytic digestion peptides from FG and SG groups were analyzed by the Thermo Scientific Orbitrap Elite combined mass spectrometer and the same batch of larvae as the test samples.We built a predicted library of polypeptide by the transcriptome sequencing data to search the mass spectrometry information.With the Max Fold Change ? 2 and Anova p-value ? 0.05 as the 2limit filters,8 major proteins in FG group expressed significantly higher than the SG group and 104 major proteins in FG group expressed significantly lower than that in the SG group.Three regulation networks of controlling the growth rate of Macrobrachium nipponense obtained by combination analysis by the mass spectrometry and high-throughput sequencings.The results of combination analysis showed that the different growth rate between FG and SG is due to the difference level of transcription as well as the difference in metaboliccatalysis enzymes and it essentially control by mi RNAs.Three networks were found for controlling the growth of the Macrobrachium nipponense as follows:Networks 1: sha-mi R-21 regulates the target gene CFL and act on the actin depolymerizing factor?cofilin/actin-depolymerizing factor?,predicted it is as the role of positive regulator in muscle development of Macrobrachium nipponense.Networks 2: sha-mi R-21 and hsa-mi R-21-5p all control the target MCCC1 homologous gene and act on the beta-methyl butene acyl Co A carboxylase?methylcrotonoyl-Co A carboxylase?,and adjust the valine,leucine and isoleucine degradation pathways,predicted it is as the role of positive regulator in the utilization of amino acids and promote the growth of Macrobrachium nipponense.Networks 3: Newly discovered mi RNA novel₂2 controls the target Tspst homologous gene and act on the proteasome subunit beta type-7.It regulates the ubiquitin proteasome pathway.We predicted it as the role of positive regulator in control protein degradation rates and be beneficial to the anabolism of Macrobrachium nipponense in the growth.The sha-mi R-21 regulated network 1 and 2 at the same time,associated with the amino acids metabolism way of the growth network in muscles.It acts a crucial role to regulate the difference growth rate in the full-sib family.This study constructed the full-sib family of oriental river prawn successfully.From the histology,m RNA,mi RNAs and protein levels,detaily studied the reasons for differences growth rate basis on molecular regulation by genes of full-sibs family of Macrobrachium nipponense eliminated the environmental impact.Obtained three networks of controlling growth of Macrobrachium nipponense,and provided a reference to cultivate new pedigree of fast-growing Macrobrachium nipponense.