A Comparative Transcriptomic And MicroRNA Omics Analysis During Embryogenesis And The Function Study Of Immune Genes From Cherax Quadricanatus

The red claw crayfish（Cherax quadricanatus）is an emerging and important commercial aquatic animal and is also a potential biological model in crustacean biology due to its biological characteristics.Embryo development is the starting point of ontogeny and the quality of embryo development is one of the important factors affecting the constitution of crayfish.The pathogen is another important factor affecting crayfish culture.The innate immune system is a natural defense system for crustaceans to remove and kill pathogens.The research on embryo development and innate immunity of crayfish is of great significance to enrich the development pattern and immunity research of crustaceans,and also provides important guidance for disease prevention of crayfish culture.In the present study,the transcriptome and microRNA omics techniques were used to study the different embryonic development stages of C.quadricanatus.Meanwhile,based on the transcriptome data,gene cloning and functional verification of C-type lectin and Kazal-type serine protease inhibitor involved in innate immunity were conducted.The main research results are as follows:1、A comparative transcriptomic analysis of C.quadricarinatus embryogenesisHigh-throughput Illumina sequencing technology was used to investigate transcriptome profiles of three embryonic development stages of C.quadricarinatus and further analyzed genes related to development.A total of 49,436 unigenes were annotated and clustered,of which 13,727 were annotated in the NR database,5,087were classified according to GO annotation,and 2,735 were related to 189 KEGG pathways.Furthermore,a total of 6,658 differentially expressed genes（DEGs）were identified by differential analysis of gene expression among different embryonic development stages.A total of 3,300 unigenes were identified as DEGs between the eye pigments forming stage（EP）and prepare-hatching stage（PH）,of which 1,595 were annotated to the database;5,211 unigenes were identified as DEGs between EP and larvae（L）,of which 2,540 were annotated;1,262 unigenes were identified as DEGs between PH and L,of which 680 were annotated.This study focused on differentially expressed genes related to morphological or trait characteristics,signaling pathways and immune system.Ato,Slit and Robo genes related to neurogenesis,Cnc and mlpt genes related to body segmentation and Apontic,Ey,Pax6 and So genes related to eye development were all largely expressed in the EP stage,indicating that EP stage is a critical period for the formation of nervous system,body segmentation and eye.Projectin genes related to the formation of appendages were highly expressed in EP and PH phases,indicating that EP and PH phases were the main stages of appendage development.The signaling pathways such as Hedgehog,MAPK,Wnt,TGF-beta,and Notch,were higher expressed in the EP stage than in the other two stages,suggesting that EP has more active biological process than in the latter two stages.During the three embryonic development stages,immune genes related to peroxisome,phagocytic and lysosome were abundant,suggesting that C.quadricarinatus had formed a relatively complete immune system in the embryonic stage,and phagocytes played a major role.2、Identification and profiling of microRNAs during embryogenesis in C.quadricarinatusMiRNAs and their target genes were identified during three embryonic developmental stages of C.quadricarinatus.Nineteen known miRNAs and 331 novel ones belonging to 50 miRNA families were obtained.A total of 113 differentially expressed miRNAs were identified,and 2,575 target genes were predicted,among which 1,257 were annotated.Additionally,63 target genes of 9 miRNAs in C.quadricarinatus were found to be related to embryonic development.For example,mi R-10 and its target genes may regulate nervous system development and body segmentation.Mi R-2788 may regulate cell proliferation to impact embryonic development.Moreover,mi R-28（target gene tutl）,mi R-50（target gene fbx5）and mi R-1260b（target gene sif）may co-regulate eye development of C.quadricarinatus embryonic.A regulatory network between miRNA and its negatively regulated target genes was analyzed.These miRNAs together with their target genes constitute a network for regulating the development of tissues and organs in the embryo of C.quadricarinatus.3、Molecular cloning and expression of C-type lectin（CqCTL）from C.quadricarinatusThe first C.quadricarinatus C-type lectin gene（designated CqCTL）was cloned and characterized based on the results of transcriptome.The complete c DNA sequence of CqCTL contained an open reading frame（ORF）of 543 bp,which encoded a protein of 180 amino acids.A carbohydrate recognition domain（CRD）containing four conserved cysteines(Cys⁴⁸,Cys⁵⁹,Cys⁷⁶ and Cys¹⁷⁷)and the EPD(Glu⁸⁰-Pro⁸¹-Asn⁸²)and QPD(Gln¹⁴⁶-Pro¹⁴⁷-Asn¹⁴⁸)motifs were identified in the deduced amino acid sequence of CqCTL.CqCTL exhibited high similarity with previously identified C-type lectins from other species.The mRNA transcripts of CqCTL were ubiquitously detectable in all the tested ten tissues,with the highest expression level in hepatopancreas.The c DNA fragment encoding the mature peptide of CqCTL was recombined into p ET-32a（+）with N-terminal Trx-,His-,and S-tags fused in-frame and expressed in Escherichia coli BL21（DE3）p Lys S,and an apparent MW of 37 k Da soluble CqCTL was obtained by using affinity chromatography method.4、Molecular cloning,expression and functional study of Kazal-type serine protease inhibitor（CqKPI）from C.quadricarinatusKazal-type serine protease inhibitor（designated CqKPI）was cloned and identified based on the results of transcriptome in C.quadricarinatus.The ORF of CqKPI contained 405 nucleotides and encoded a protein of 134 amino acids.CqKPI had two Kazal domains,both of which were composed of 44 amino acid residues,with the conserved amino acid sequence C-X₃-C-X₅-PVCG-X₅-Y-X₃-C-X₆-C-X₁₂-C,that is,each Kazal domain contained 6 conserved cysteines,which could form the conformation of three pairs of disulfide bond stabled Kazal domain.The P1 amino acids of the Kazal domains were Ser⁴³ and Lys⁹¹,respectively,suggesting that CqKPI may inhibit trypsin and elastase.CqKPI exhibited high similarity with previously identified Kazal-type serine protease inhibitors from other species.The mRNA transcripts of CqKPI were ubiquitously detectable in all the tested ten tissues,with the highest expression level in hemocytes.The recombinant CqKPI was successfully expressed in E.coli and purified by Ni-NTA chromatography and size-exclusion chromatography.The recombinant CqKPI could bound to Bacillus hwajinpoensis,Staphylococcus aureus,Vibrio parahaemolyticus,and Candida albicans,and inhibit the growth of B.hwajinpoensis and C.albicans.These results indicate that CqKPI may participate in the innate immune system of C.quadricarinatus by binding pathogenic bacteria and interacting with serine protease of pathogens to help C.quadricarinatus resist the invasion of pathogens.