| The pangolins were the only mammal that grows scales all over its body and were also listed on the IUCN Red List of Threatened Species.As the most representative feature of pangolins,the scales not only possessed excellent physical properties,but also had medicinal values such as activating blood circulation and lactation.However,studies on scale formation were still at the stage of morphological observation and exploration of physicochemical properties,and the identification of molecular structure was lacking.1.Chromosomal genome assembly of Malayan pangolin and Chinese pangolin.The whole genomes of the two pangolins were high-throughput sequencing by using the Nanopole sequencing platform technology,and were de novo assembled.Further,we used illumina sequencing data to correct assembly errors and Hi-C technology was used to assemble genome to the chromosome-level scaffolds.The total length of the chromosome genomes of Malay pangolins and Chinese pangolins was 2.44 Gb and 2.53 Gb.Respectively,the contig N50 sizes of the genomes were 15.81 Mb and 13.97 Mb,and they both contain 19 pseudochromosomes.With the transcriptome sequencing data,gene annotation was performed by de novo prediction and homology-based prediction,and then yielded 21363 genes of the Malayan pangolin and 20428 genes of the Chinese pangolin,respectively.The integrity of core genes reached 96.20% and 95.70%by BUSCO software.2.Pangolin gene family analysisIn order to uncover the genes of important structural proteins involved in the formation of the epidermis and its appendages,we annotated artificially genes of the epidermal differentiation region(EDC),keratin(KRT)family and keratin associated protein(KRTAP)family.Multiple genomic structural variation in the epidermal differentiation region of the pangolin were identified in a multi-species comparative genomic covariance study,including a family of small proline-rich protein genes(SPRRs)involving in UV resistance,which were lost during the evolution of the pangolin and may be a key reason for the nocturnal activity of the pangolin.Gene expression analysis revealed that the pangolin-specific KRTAPs gene HGT underwent gene expression during evolution.3.Tissue-specific gene expression of PangolinThe transcriptome sequencing data of twenty-four tissues(hair-type skin,scale-type skin,ventral dorsal muscle,lung,heart,kidney,liver,spleen,gall bladder,large intestine,small intestine,anterior stomach,hind stomach,bladder,testis,brain,cerebellum,hypothalamus,eye,nose,tongue,blood,perianal gland)were aligned to the constructed genome to obtain the gene expression of each sample.By setting the differential expression ploidy and Z-Score screening thresholds,A total of 2086 tissue-specific genes and 1901 tissue co-expressed genes were obtained.In the tissue co-expression analysis,it was found that the expression pattern of the pangolin stomach was similar to that of the skin,and was functionally enriched in the epidermal cornification pathway,confirming the adaptive evolution of stomach structure under the toothless diet of pangolins.4.Gene expression regulatory network of pangolin scale formationTranscriptomic of scale-hair type skin and scale-hair proteomic comparative analysises showed that more types of KRT genes and KRTAP genes were more significantly expressed in the hair-type skin of the pangolin than in the scale-type skin,and that the scale-type skin mainly expressed structural genes such as KRTAP13-1,HGTs-KRTAP,KRT16 and KRT36.ATAC-seq and mi RNA-seq analyses revealed that Pangolin scale formation is largely dependent on the regulation pattern of HGT3-KRTAPs. |
