Genome-wide Transcriptome And Proteome Analysis On Important Biological Processes During The Early Development Of The Pacific White Shrimp Litopenaeus Vannamei

Penaeid shrimp, as a typical representative of crustacean, has a distinctive pattern for early development. During embryo stage, it gets through the journey from zygote to cleavage embryo, blastula, gastrula, limb bud embryo and larva in membrane. After hatching from membrane, it steps into larva stage which includes nauplius, zoea, mysis and postlarvae. Both its morphological and physiological features change dramatically along with metamorphosis in this period. The researches on early development of penaeid shrimp will provide insight into crustacean evolution and development modularity and also will have considerable significance for shrimp larvae aquaculture. For now, many studies are focused on nutriology of larvae shrimp while little is known about the molecular mechanism of shrimp development. Omics technologies provide a brand new way for acquiring the expression patterns of genes and proteins comprehensively. In this thesis, we use genome-wide transcriptome and proteome analysis for understanding the biological processes during the early development of Pacific white shrimp Litopenaeus vannamei. The main progresses are as follows:1. The transcriptomes of embryo, nauplius, zoea, mysis and postlarvae were sequenced with Illumina sequencing technology. The reads were assembled and clustered into 66,815 unigenes. 37,292 of which have annotations in Nr, Nt, Swiss-Prot, GO, COG or KEGG databases. The differentially expressed genes(DEGs) between adjacent developmental stages were identified. GO term and KEGG pathway enrichment analysis showed that DEGs from embryo to nauplius were significantly enriched in hormone induced programmed cell death process which related to histolysis and reconstruction; DEGs from nauplius to zoea were significantly enriched in pancreatic secretion and nutrient metabolic pathway which related to food digestion and absorption; DEGs from zoea to mysis were significantly enriched in myosin Ⅱ filament assembly and organization which related to muscle development; DEGs from mysis to postlarvae were significantly enriched in chitin or polysaccharide metabolic process which related to exoskeleton reconstruction. Twenty early developmental samples of L. vannamei were also sequenced for acquiring a more precise gene expression pattern. By hierarchical clustering analysis and principal components analysis, 20 samples were divided into 3 groups. Group 2 which was composed of larva in membrane and nauplius was inferred as a crucial transition period for development.2. Two quantitative proteomic approaches were applied to analyze the proteins related to early development of L. vannamei. Using a 2D-DIGE system, we compared the proteome differences at five stages: zygote, blastula, gastrula, limb bud embryo and nauplius. 90 differentially expressed protein spots were identified according to MALDI-TOF-MS/MS. Based on the i TRAQ method, a total of 2560 proteins were detected. The differentially expressed proteins across eight early developmental stages were also analyzed.3. Alignment and comparative analysis were conducted among the acquired gene set from our study, EST data set from L. vannamei and functional gene sets from other arthropods. We then mapped our Illumina sequencing data to the genome, and analyzed the characteristics of functional genes. A series of exon junctions and gene arrangement information were also obtained.4. The molecular basis of morphological change during early development of L. vannamei were analyzed from the perspective of muscle development and cuticle reconstruction. For muscle development, the vital regulatory signals twist and mef2 were expressed from limb bud stage, while constitutive proteins projectin were expressed from larva in membrane stage. For cuticle reconstruction, we found that chitinases which are responsible for molting and cuticle proteins which constitute the exoskeleton were highly diversified. We also inferred that the calcification of exoskeleton occurred at postlarvae stage according to the expression pattern of calcified cuticle proteins.5. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during early development of L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Most of the digestive enzymes kept a relatively low expression level at embryo and nauplius stages. Their expression increased sharply at zoea stage. After the expression of peptidase decreased at mysis stage, the expression of digestive enzymes kept stable at postlarvae stage. Their diversity appeared along with development and multiple alignments of amylase genes inferred the different expression preference between larvae and adults.6. The formation of shrimp immune system were also discussed according to our RNA-Seq data. We found three initiating periods according to the expression pattern of immune related genes: the first initiating stage was zygote which inherit from maternal immunity; the second was limb bud embryo when immune-related pathway became activated; the third was zoea when massive immune effectors appeared. The regulatory pathway for hematopoietic differentiation was identified. The expression pattern showed that embryo hematopoiesis happened at limb bud embryo stage, while larva hematopoiesis happened at zoea stage. We identified ten Toll genes according to the early developmental transcriptomes of L. vannamei and analyzed their domain and structure. Their tissue distribution and expression pattern when infected with WSSV were analyzed in adult shrimp.