Studies On The Roles Of Gene Family Expansion And Alternative Splicing Events In The Environmental Adaptation For The Shrimp Litopenaeus Vannamei

Penaeid shrimp,belonging to Decapoda,Crustacea.They are representative species of aquatic animals with irreplaceable scientific value.Furthermore,as important aquaculture species in the world,shrimp presents great economic value.Deciphering the shrimp genome and accumulation of a large number of �omics� data are of great significance to clarify the special life phenomenon,illustrate the mechanism of evolutionary adaptation,analyze the evolutionary history and ecological position of shrimp,even to crustacean.What's more,it plays an important foundation for the genetic breeding of economic crustacean represented by shrimp.In this paper,the relationships between the expansion of three gene families and alternative splicing events of the whole genome and the environmental adaptation were systematically analyzed based on the genomic data and transcriptomic data of L.vannamei.The researches will not only lay an important foundation for clarifying the molecular mechanism of environmental adaptation of shrimp and other crustaceans,but also have some guiding significance for the regulation of culture environment and ecology of shrimp.The major results are as follows:1.Studies on the structure and function of Actin and Myosin heavy chain(MYH)gene families in L.vannamei.In order to adapt to benthic life,the well-developed abdominal muscle system enables shrimp to escape from the predators quickly.Actin and MYH are both major muscle constituent proteins.Combined with genomic and transcriptomic data,37 Actin and 16 MYH genes were characterized in shrimp.Based on the sequence structure,functional domain and phylogenetic tree,they could be classified into four types: fast muscle type,slow muscle type,heart type and cytoplasmic type.Compared with other related species,fast type Actin,slow type Actin and fast type MYH were significantly expanded in shrimp genome.The feature of high sequence similarity and clustered distribution in the genome indicated that they might expand through tandem replication.The temporal and spatial expressions of different types of Actins and MYHs were significantly different in shrimp.The fast type Actin and MYH were highly expressed in the abdominal muscles,the slow type Actin and MYH were highly expressed in muscle-containing tissues,the heart type Actin and MYH were highly expressed in heart,and the cytoplasmic type Actin and MYH were highly expressed in all detected tissues.Most Actins and MYHs began to be expressed from limb bud stage to nauplii in membrane stage,and the expression increased gradually with the development of larvae and the enhancement of motor ability.These results suggested that shrimp muscle system mainly developed at zoea and mysis stage.Through in situ hybridization analysis,we found that slow type Lv Actin SSK5 and Lv MYH5 genes were both mainly expressed in the superficial ventral muscle and pleopod muscles,which are regarded as slow muscle.Therefore,the expanded fast-type Actin and MYH may explain the well-developed muscle system of shrimp,which makes them have excellent explosive power.The expanded slow-type Actin make them have long-term swimming ability.The cooperation of explosive power and swimming ability enable the shrimp to escape from predators quickly.2.Analysis on the structure and function diversity of juvenile hormone esterase-like carboxylesterase(CXE)gene family in L.vannamei.Shrimp usually experiences about 50 molts during their lifetime.Such frequent molting provides a guarantee for rapid growth of shrimp.Juvenile hormone esterase(JHE)plays an important role in the early development and molting process in insects.CXE gene in crustacean is regarded as a homologue with JHE gene,but its function in the complex molting pathway of shrimp is unclear.Based on genomic and transcriptomic data,21 Lv CXEs were characterized in L.vannamei,which was one of significantly expanded gene families in shrimp.All Lv CXEs have a conserved triplet catalytic site and a characteristic Gx Sx G motif.Among them,13 of 14 Lv CXEs containing a GESAG motif were clustered in the phylogenetic tree,indicating that they were specifically expanded in shrimp.Most of Lv CXEs were highly expressed in hepatopancreas,intestine and ovary,which were the major sites for methyl farnesyl(MF)degradation.It suggested the potential functional relationships between Lv CXE and MF.Among them,10 Lv CXEs,including 7 genes containing GESAG motif,were highly expressed from zoeae stage to post-larval stage.Totally 16 Lv CXEs,including11 genes containing GESAG motif,presented molt-dependent expressions,with the lowest expression at pre-molt D3 stage during the molt cycle.When injecting OTFP,a specific inhibitor of JHE in insects,at the pre-molt stage of shrimp,they failed to molt and died finally.Furthermore,the expression levels of four key ecdysone responsive transcription factors,including Lv E75,Lv Br-c,Lv Hr3 and Lv Ftz-f1,were down-regulated after OTFP injection.These results suggested that the important role of Lv CXEs in shrimp molting process.In conclusion,the functional diversity of expanded Lv CXEs plays vital roles in the embryo development and molting process of shrimp.3.Studies on alternative splicing events and functional diversity in the whole genome of L.vannamei.Alternative splicing is one of the main ways to regulate gene expression and generate proteome diversity.However,research on alternative splicing of shrimp is very limited.Combined with genome and multiple transcriptome libraries,38,781 alternative splicing events of 9209 genes were identified in shrimp.The ratio of exon skipping(ES),alternative 3' splice sites(A3SS),alternative 5' splice site(A5SS),intron retention(RI)and mutually exclusive exon(MXE)was 13.35%,11.43%,10.8%,6.77%,2.36%,respectively.Among all transcripts,92.93% used the canonical GT-AG dinucleotides at the 5' and 3' sites,followed by GC-AG and AT-TC.Compared with non-AS genes,AS genes were longer and contained more and shorter exons and longer introns.The functions of non-AS genes were mainly related to DNA integration,DNA metabolic process,nucleic acid metabolic process and neurological system process,while multi-AS genes were mainly responsive to biotic and environmental stress.According to the difference in the increasing trend of the splicing form,AS genes can be divided into type ? genes and type ? genes.Type ?genes possess only one transcript in the control group but they are alternatively spliced in the experimental group of stressed libraries.Type ? genes possess specific AS events in the experimental group under stressed conditions compared with the control group.After injection of white spot syndrome virus(WSSV),Vibrio parahaemolyticus and Staphylococcus aureus,the total number of AS events in the whole genome increased significantly,and the number of various types of AS increased overall.The function of type ? genes was similar and mainly related to substance metabolism or energy metabolism,which plays a role in maintaining the basic metabolic balance and ensuring the material and energy supply.The function of type ? genes was mainly related to immunology,but it was different for different pathogens.In detail,the function of type ? genes was related to NOD-like receptor signaling pathway in WSSV group,ECM receptor interaction and intestinal immune network for Ig A production pathway in V.parahaemolyticus group,ECM receptor interaction,shigellosis,pathogenic Escherichia coli infection,bacterial invasion of epithelial cells pathway in S.aureus group.These results showed that the pathogen could be eliminated by changing the AS pattern of immune related genes when shrimp was infected by microorganisms.What's more,the expression levels of seven splicing factors were up-regulated in above three groups,suggesting some similarity of AS regulation during microbial challenge.Additionally,AS also plays an important role under the stress of physical environmental factors in shrimp.The total number of AS events also increased significantly under low salinity stress.Type ? genes were mainly enriched in peroxisome and purine metabolism process,and type ? genes were mainly related to cation binding,ion binding and betalain biosynthesis process.These processes could maintain the balance of osmotic pressure in shrimp.What's more,heat stress induced the specific AS events of ES,A3 SS and A5 SS,but repressed RI.The AS patterns of genes related to RNA binding,binding and nucleic acid binding were changed to amplify the downstream regulation of AS and transcription in gill under heat stress.The AS patterns of genes related to ubiquitin-protein transferase activity and nuclear ubiquitin ligase complex process were changed to remove misfolded proteins in hepatopancreas.The above results indicated that AS plays a crucial role in response to external environmental stress in shrimp.In shrimp genome,the expansion of gene families and the increase of AS events enrich the diversity of gene composition and expression,and then promote the functional diversity of genes.The significant expansion of Actin and MYH gene families promoted the evolution of fast type and slow type muscles,and enhanced the ability of shrimp to escape from predators quickly.The significant expansion of CXE gene family provided precise regulations for frequent molting in shrimp.The abundant AS events not only increase the structural and functional diversity of transcripts and proteins,but also enable shrimp to acquire strong environmental adaptability.It reflects a unique response and adaptation mechanism in shrimp under the stimulation of biological factors or physical environmental factors.In conclusion,gene expansion and AS are both important ways for shrimp to adapt to the environment.