Rna-seq Analysis, Immune Gene Discovery And EST-SSR Markers Development Of Andrias Davidianus

The Chinese giant salamanders Andrias davidianus is an important species in the processof evolution and it also has the high economic value in china. Due to long-term neglect ofresource protection, the wild resources are being dramatically declined, making it anendangered species. With the success of artificial breeding and the expansion of the size of A.davidianus farming, the giant salamander resources got rapid recovery. However, there are nosufficient transcriptomic and genomic data in public databases for understanding of theimmunologic mechanism, disease resistant mechanism and genetic background. Thus itcannot fundamentally solve the problems in the process of artificial breeding for diseaseprevention and control and wild giant salamander resources protection.This study we used Illumina paired-end sequencing to characterize the transcriptome ofthe skin and spleen. Then, we used the transcriptome data to analysis the differentiallyexpression between the skin and the spleen, and obtained a number of specifically expressedimmune genes in skin and spleen. At the same time, we developed a batch of new EST-SSTmolecular markers, obtained the following results:In this study, more than40million sequencing reads of spleen and skin tissues weregenerated using Illumina paired-end sequencing technology. De novo assembly yielded87,297unigenes with a mean length of734bp. Based on sequence similarity search withknown proteins, a total of38,916(44.58%) genes were identified. Out of these annotatedunigenes,34,972and28,608unigenes were assigned to Nr and Swiss-prot database,respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathwaydatabase (KEGG) indicated that12,774unigenes were mapped to243KEGG pathways, and995were assigned to immune system pathway.Tissue-specific expressed analysis indicated that a total of225immune gene specificexpressed in spleen, to the contrary, a total of299immune gene specific expressed in spleen.Among these specific expressed unigenes, some sequences were identified be related toresponse to external stimulus, immune response and inflammatory reaction, such as fucolectin, β-defensins, lymphotoxin beta and immunoglobulin light chain type III. Real timequantitative PCR (qRT-PCR) analysis was conducted for nine tissue-specific genes which weselected for validation, suggesting that they were significantly higher expressed in skin andspleen than in other tissues, respectively; and the expression of these genes had no directrelationship with age.We used MISA software to screening the EST-SSR molecular markers from the unigenesdatabase of A. davidianus. After screening, we obtained5489unigenes containing7,961EST-SSR loci from16,599unigenes, accounted for33.06%of the screening of unigenes.Among the EST-SSR loci, most of them (81.28%) being mono-nucleotide SSRs, followed bydinucleotide, trinucleotide and tetranucleotide, pentanucleotide, no hexanucleotide werefound.In situ hybridization results showed, the positive signals of galectin-3were found in theepidermis of the skin. Weak positive hybridization signals also appeared in the dermal layerof mucus gland glandular epithelial cells and granule cells. The positive hybridizationsignals of SLC40A1in red pulp of the spleen were obvious than in the white pith, positivesignals also appeared in the integument of spleen.Our study indicates that Illumina pair-end sequencing is an efficient, convenient, highreliability method to characterize the whole transcriptome and discover functional gene. Ourdata represent the comprehensive sequence resource of A. davidianus. These gene resourcescan provide a basis for future research on immunologic mechanism and disease resistantmechanism of A. davidianus. The skin and the spleen play important roles in the immunedefense system of giant salamander, but differences between the key immune factorsinvolved in this process, which demonstrates the immune defense mechanisms of the skinand the spleen is distinct; In order to better adapt to the environment, the immune system hasbeen developed completely before A. davidianus becoming one year old. Using thetranscriptome database can obtain large number of EST-SSR molecular markers can providetheoretical basis for studying the population genetic structure, molecular geneticcharacteristics and molecular marker assisant selection.