Solexa-Sequencing Based Transcriptome Study Of Plaice Skin Phenotype In White Rex Rabbit

Rex, as a typical skin rabbit, is loved by people because of its unique coat. In recent years, people found that some of the rabbit appeared plaices in the abdomen, buttocks and extremities during the production, which we called Plaice Rabbit. The key different of two phenotype rabbits was the area of skin. For skin with animals, the area of hide and fur quality are important factors in affecting their economic benefits. And the Plaice Rabbit has greater skin area and same quality of skin, so it provide a new direction in the farming of rabbit.As a new generation of high-throughput sequencing, transcriptome could study the function and structure of gene from the overall level. Based on the Illumina platforms, transcriptome sequencing were able to detected in any species on the level of single nucleotide. Transcriptome technology can discover unknown and rare transcripts in analyzing the structure and the expression levels of transcripts. Compared to traditional microarray platform, transcriptome sequencing can provide a more accurate digital signal, higher throughput and broader detection range. So it is a powerful tool for in-depth study of the current complexity of the transcriptome.In this study, plaice rabbit(experimental group) and un-plaice rabbit(control group) were sequenced by RNA-Seq technology. Relating to the functional analysis of the sequencing results, screening genes which associated with the growth and development of rabbit skin.Under the using of Trinity, the method of pair-ends repeated was selected for de novo assembly after merged effectively reads. Two phenotype skin samples were sequenced a total of 377,618 Unigenes, and the average length of Unigenes is 680 nt. In all these Unigenes, the longest sequences 20,293 nt.After got Unigenes, we blasted sequences to some databases, such as NR, Swiss-Prot, KEGG, and KOG, on the basis of annotation in these databases, we analyze the function of Unigene which were known.85.5931%sequences could not blast to any database, because of the incomplete whole genome information of rabbit. According to the NR database, in total of 42,895 Unigenes were blasted to different GO Term and grouped into three categories:Biological Process(P), Cellular Component(C) and Molecular Function(F). By KOG, all the 60,593 Unigenes were classified into 25 categories. And in KEGG Pathway database, we known 13,431 Unigenes were blasted to 399 different pathways. The most of Unigenes were annotated PI3K-Akt pathway(ko04151). This signal pathway was play an important role during the growth and development in rabbit skin.Analysis samples of two different phenotypes, taking P-Value≤ 0.05 and Iog2|foldchange|> 1 as the criteria of selection,we got 308 significant differentially expressed genes.118 Unigenes of them were up-regulation and 190 were down-regulation in Plaice rabbit samples. Further analysis showed that 99 Unigenes could found information in GO Term and 29 were annotated to KEGG Pathway. Another 209 Unigenes could not be blasted to any databases called novel gene. Among the 209 Unigenes,33 only expressed in Un-plaice rabbit and 56 merely expressed in Plaice rabbit.In addition, qRT-PCR was opt to validate the results of sequencing.7 target genes were designed specific primers by oligo 7.0, using AACt method to calculate the relative expression of Plaice and Un-plaice phenotype samples. The results showed the consequence of qRT-PCR and transcriptome sequencing was consistent. From the differentially expressed genes, we identified genes associated with the development of rabbit skin preliminary, and conducted further experiments.Candidate gene MYL12B was important components of the cytoskeleton. As myosin regulatory light chain, MYL12B play a role in the dynamics of many cells by adjusting phosphorylation and dephosphorylation of the light chain. In this study, we cloned CDS sequence of MYL12B, and compare the homology of amino acid sequence encoded by CDS with other 12 species:human, chimpanzee, green monkey, mouse, etc. Result showed that, the gene was high similarity with mammals except horse, up to 94%..With birds and amphibians, the sequence also have high homology. Bioinformatics analysis revealed that the protein is an unstable structure and Hydrophilic protein and rich in phosphorylation and glycosylation sites. In two phenotype rabbits, MYL12B has no difference of the CDS sequence. The difference in foldchange maybe dependent on the mechanism of MYL12B.Chosen target gene LAMB3 for PCR-SSCP, the result of acrylamide gel electrophoresis showed that there was 1 potential mutation sites in 8th exon. Through statistical analysis, the mutation in the skin samples changed the structure of LAMB3 protein, and maybe made the function also changed. This is perhaps one of reasons causing the Plaice and Un-plaice phenotype.The rabbit transcriptome profiling data provide new insights in understanding the molecular mechanisms underlying rabbit skin growth and development. Determined on relation genes with the skin growth and development of plaice rabbit. And study the function and location of genes preliminary. Also, to further study the gene provides the basis for the molecular and protein levels of function and mechanism of action.