Transcriptome Profiling Analysis And Transforming Upon Acute Overstocking Stress In Larimichthys Crocea

The large yellow croaker,Larimichthys crocea is an important marine fish in China with a high nutrient and economic value.In recent years,with the technological breakthroughs of artificial breeding and large-scale farming,the aquaculture industry of L.crocea presented the trend of rapid development,leading to a enormous social and economic benefits.However,knowledge of molecular genetics and transcriptome sequences L.crocea have not been thoroughly analyzed,which would be unfavorable to identify functional unigene and develop marker-assisted selection.In this work,we sequenced the transcriptome in L.crocea liver and skin tissue with Roche 454 and Illumina Hiseq 2000 sequencing platforms,repectively.After sequecing,we have assembled and annotated the L.crocea transcriptome,and identified massive SSR markers from it.Finally,we obtained amount of differentially expressed genes(DEG)by comparing the skin transcriptomes.Detailed results and conclusions are as follows.1.We have sequenced the L.crocea liver tissue by Roche 454 pyrosequencing technology and obtained 316,523 raw reads.All clean reads were then assembled into 93,801transcripts,containing 4,993 isotigs and 88,808 singletons.Average length of the assembled transcriptome was 332 bp and size of it was 48.7 Mb.Of them,38,856(41.4%)transcripts were successfully annotated in nt,nr,Swiss-Prot,InterPro,COG,GO and KEGG databases.Based on the annotation information,2,691 unigenes were related to growth and 474unigenes were associated with immunity,repectively.This work would provide valuable genetic resources for future gene functional analysis and molecular maker development of L.crocea.2.Additionally,a total of 6,391 simple sequence repeats(SSRs)were identified from the assembled transcriptome,among which 4,498 SSRs had enough flanking regions to design primers for polymerase chain reactions(PCR).To access the polymorphism of these markers,30 primer pairs were randomly selected for PCR amplification and validation in 30individuals,and 12 primer pairs(40.0%)exhibited obvious length polymorphisms.The number of alleles for the 12 SSR loci varied from 2 to 10,with an average of 5.5 alleles.The observed heterozygosity(H_O)ranged from 0.100 to 1.000,while the expected heterozygosity(H_E)varied from 0.097 to 0.885.Polymorphic information content(PIC)ranged from 0.090to 0.856.This demostrates SSR markers identified from L.crocea transcriptome are fairly stable and exhibit high polymorphism among individuals.3.Acute overstocking is a comprehensive and common stress that fish in aquaculture farm might undergo during water renewing,feeding,shipping and even storing.However,the gene expression profile of fish under the overstocking stress is extremely little reported.In this work,we studied the transcriptome profile of abdominal skin of L.crocea overstocked for 0h(control),0.5h and 2h by the Illumina HiSeq 2000 platform.In total,758 and 1,912 differentially expressed genes(DEGs)were identified when the gene expressional levels were compared between 0h and0.5h(0h-0.5h)and 0h-2h,respectively.Among them,397 DEGs were up-regulated,361 DEGs were down-regulated(0h-0.5h);In the comparision of 0h-2h,1,016 DEGs showed up-regulated,896 DEGs showed down-regulated.Functional analysis in GO and KEGG annotations revealed that genes related to immune responses,energy metabolism,cell differentiation and apoptosis were greatly enriched.Important genes contributing to these biological functions,including ATF3,LECT2,ADM,IL-6/INF-α,PAH and TRα,were obviously regulated to avoid cerebral inflammatory injury and to maintain energy balance during the stress.The detailed transcriptome profile analysis also revealed regular expression patterns of DEGs for stress durations,indicating the elaborate gene regulation networks underlying the molecular responses to the overstocking stress.Our transcriptome analysis demonstrated systematic and complicated regulatory cascades upon the overstocking stress in large yellow croaker,which providing important gene expression profiles to study stress response on a highly commercially important marine fish species.