Abstract:Homeobox genes are ubiquitous in eukaryotic genomes and can be transcribed into a homeodomain of approximately 60 amino acids. Hox genes are currently the most studied class of homeobox genes. They are usually clustered on chromosomes and play important regulatory roles in embryonic development, cell differentiation, body pattern determination, and tissue and organ formation of organisms. In this study, the bioinformatics methods were used to identify the Hox gene family based on the genome and transcriptome data of Procambarus clarkii to comprehensively understand the distribution, evolution, and function of the Hox gene family in the genome of P. clarkii. The sequence structure, protein physicochemical properties, motif composition, phylogenetic characteristics, and adaptive evolutionary features were analyzed. The expression of Hox genes in different developmental stages of P. clarkii and different tissues of adult crayfish were studied by fluorescence quantitative PCR to explore the possible biological function of Hox genes and provide a theoretical basis for analyzing the morphological development mechanism of P. clarkii. The results showed that eight Hox genes on the same chromosome were identified in the genome of P. clarkii, namely lab, pb, Dfd, Scr, Antp, Ubx, abd-A, and Abd-B. Hox proteins are composed of a YPWM-containing motif and a highly conserved homeodomain, and the homologous domains of Hox proteins are highly similar among different species. The physicochemical properties analysis of Hox proteins showed that the amino acid sequence length of Hox proteins ranged from 278 to 608, the molecular weight ranged from 31 130.52 to 64 355.33 Da, and the isoelectric point ranged from 6.73 to 9.51. All Hox proteins are hydrophilic, unstable proteins that are located in the nucleus. Motif composition analysis showed that the Hox genes clustered into the same cluster were conservative. Motif1 was located in the homologous domain and existed in all Hox genes. Conserved domain analysis showed that Hox proteins contained the homeodomain, and abd-A protein also contained the Abdominal-A domain. Genomic collinearity analysis between species showed collinearity between multiple chromosomes of P. clarkii and Portunus trituberculatus and the Hox genes (pb, lab, and Dfd) on the LG30 chromosome where the Hox gene cluster of P. clarkii was located, and the Hox gene on chromosome 49 of P. trituberculatus showed collinearity. Phylogenetic analysis showed that different Hox gene family members were clustered into one branch. On the branches of pb, Dfd, Scr, lab, and Antp genes, crayfish, and crabs were first clustered into one branch and then clustered into another with prawns. On the branches of abd-A and Abd-B genes, crabs and prawns were first clustered into one branch and then clustered into another branch with crayfish. On the branch of the Ubx gene, the crayfish first clustered with the prawns and then clustered with the crabs. This indicates that different members of the Hox gene family have different phylogenetic histories in shrimp and crab species. The adaptive evolution analysis showed that the Hox genes of crustaceans were mainly subjected to purification selection, and the evolutionary rates of Abd-B and Dfd genes were significantly different between P. clarkii and other crustaceans (P<0.01). Positive selection sites with posterior probability greater than 0.95 were detected in pb and Ubx genes. The relative expression of Hox genes in different developmental stages of P. clarkii showed that except for abd-A and Dfd genes, the expression levels of other Hox genes were the highest in the zoea stage, and all showed a trend of increasing first and decreasing subsequently. Among them, abd-A, Antp, and Ubx genes were lowly expressed in the gastrula and nauplii stages and then highly expressed. The distribution of Hox genes in different tissues of P. clarkii showed that the relative expression levels of different Hox gene family members vary across various tissues in adult crayfish. In addition to the lab gene, the relative expression of Hox genes in different tissues of female crayfish was generally higher than that in different tissues of male crayfish. In summary, this study used bioinformatics methods to perform genome-wide identification, protein structure analysis, amino acid sequence analysis, physical and chemical properties analysis, subcellular localization prediction, collinearity analysis, phylogenetic analysis, adaptive evolution analysis, and expression analysis of Hox genes in early developmental stages and different tissues of adult crayfish. The results showed that eight Hox genes clustered on the same chromosome were identified in P. clarkii. The amino acid sequences and protein physicochemical properties of different Hox genes differed. However, except for the Abd-B gene, they all had a YPWM-containing motif and a highly conserved homeodomain, and the protein structure was similar. Phylogenetic analysis showed that different members of the Hox gene family had different phylogenetic histories in shrimp and crab species. The adaptive evolution analysis showed that Abd-B, Dfd, pb, and Ubx genes had different evolutionary rates among crustacean species. The expression analysis of Hox genes in the early developmental stages and different tissues of adult crayfish showed that lab and Dfd genes may be involved in the differentiation of the head, chest, and abdomen of P. clarkii in the early stage of embryonic development. The expression differences of abd-A, Ubx, and Antp genes between shrimp, crayfish, and crabs may be the potential factors causing the morphological differences in their tails. This study provides a reference for further functional research and analysis of the morphological development mechanism of P. clarkii.
