Bioinformatics-Based Molecular Evolutionary Analysis Of TLP, SBP-box, CPP-like, Cystatin And HAK Gene Families In Rice

The molecular mechanism for genesis, differentiation of gene families was the important content in the field of molecular evolution. With the completeness of sequencing projects for some model organisms, more attention was put on the evolutionary analysis of gene families. There usually are more members for a gene family in plants than in animals and other organisms. This was the result of species-specific expansion of gene families in plants. Gene duplication may arise through three principal mechanisms: segmental duplication, tandem duplication and transposition events such as retroposition and replicative transposition. The main mechanism of gene duplication was segmental duplication and tandem duplication. The genes in duplicate pairs may go through positive selection and gene conversion after the duplication. Some models were developed to explain the fate of duplicated genes. Among them, nonfunctionalization, neofunctionalization, subfunctionalization and subneofuncitonalizaiton were widely accepted. In previous studies, several rounds of whole genome duplication in rice genome were reported. And this resulted in many duplicated genes. With bioinformatic analysis, the indica-japonica divergence was found occuring at the last 0.5 million years ago. Many researches consisted with the evolution of rice gene families after the sequencing of rice genomes, and the evolutionary anlyses of gene families in rice had become one of the most important subjects in the field of rice genetics. In present study, we performed the bioinformatic and evolutionary analyses of TLP, SBP-box, CPP-like, Cystatin and HAK gene families in rice. The main results showed as follows:(1) Tubby-like proteins (TLP) played an important role in maintenance and functionization of neuronal cells during postdifferentiation and development in mammals, and had been found in multicellular organisms from both the plant and animal kingdoms. We presented here a comparative phylogentic and molecular evolutionary analysis of the TLP gene family in Arabidopsis, rice and poplar. At the level of genome-wide screening, we identified 11 TLP genes in Arabidopsis, 14 in rice and 11 in poplar. Most Tubby-like proteins in plants contained both highly conserved TUB and F-box domains. Alignment of predicted protein sequences showed that there were four conserved blocks in TUB domain. Phylogenetic analysis grouped this family into three subfamilies and suggested that species-specific expansion contributed to the evolution of this family in plants. The intron distribution of this family was conserved in most members of this family, suggesting that the exon/intron structure of this family had been existent before the split of monocots and dicots. On a genome scale we revealed that the rice and poplar TLP family should have expanded mainly through segmental duplication events, rather than through tandem duplication and replicative transposition events. Co-evolutionary analysis revealed that TUB and F-box domains had possibly co-evolved during the evolution of proteins that possessed both domains. The tissue-specific expression analysis illustrated that functional diversification of the duplicated TLP genes was a major feature of the long-term evolution. Furthermore, analysis of nonsynonymous and synonymous substitution rates indicated positive and neutral selections contributed to the functional diversification of duplicated pairs.(2) SBP-box proteins are plant-specific putative transcription factors, which contain highly conserved SBP domain and could bind specifically to promoters of the floral meristem identity gene SQUAMOSA and its orthologous genes to regulate their expressions. In this study, 17 nonredundant SBP-box genes in Arabidopsis genome and 19 in rice genome were identified by using the known SBP domain sequences as queries. The phylogenetic analysis suggested that the main characteristics of this family might have been in existence before the split of Arabidopsis and rice, and most SBP-box genes expanded in a species-specific manner after the split of monocotyledon and dicotyledon. Segmental duplication events contributed mostly to the expansion of this family in two species. All the SBP-box proteins were classified into 9 subgroups based on the phylogenetic tree, where each group shared similar motifs and the orders of the motifs in the same group were found almost identical. Analysis of nonsynonymous and synonymous substitution rates revealed that the SBP domain had gone through purifying selection, whereas some regions outside SBP domain had gone through positive or relaxed purifying selection. The expression patterns of the SBP-box genes were further investigated by searching against the EST database. Results showed that the Arabidopsis SBP-box genes are expressed chiefly in flowers, leaves, roots and seeds, while those in rice mainly in flowers and callus.(3) CPP-like genes are members of a small family which features the existence of two similar Cys-rich domains termed CXC domain in their protein products and distribute widely in plants and animals but do not exist in yeast. The members of this family in plants play an important role in development of reproductive tissue and control of cell division. To gain insights into how CPP-like genes evolved in plants, we conducted a comparative phylogentic and molecular evolutionary analysis of the CPP-like gene family in Arabidopsis and rice. The results of phylogeny revealed that both gene loss and species-specific expansion contributed to the evolution of this family in Arabidopsis and rice. Both intron gain and loss were observed through intron/exon structure analysis for duplicated genes. Our results also suggested that positive selection was a major force during the evolution of CPP-like genes in plants, and most amino acid residues under positive selection were disproportionately located in the region outside the CXC domains. Further analysis revealed that two CXC domains and sequences connecting them might have co-evolved during the long evolutionary period.(4) Plant cystatins or phytocystatins are cysteine proteinase inhibitors, which exist widely in different plant species. Because these genes can kill insects and pathogens by inhibiting the digestive function of the cysteine proteinase in gut, they are believed to play an important role in plant defense against pests and pathogens. In this study, we used the cystatin domain sequences that identified from the cystatin genes in plants as queries to search for cystatin genes in both Arabidopsis and rice genomes. A phylogenetic tree was then constructed based on the corresponding cystatin proteins from Arabidopsis and rice and the conserved sequences of these proteins were analyzed. Finally we searched the Genbank EST database to get the expression information of these genes. The results showed that 7 non-redundant cystatin genes in Arabidopsis and 12 in rice were identified based the identification of the cystatin domains and combining the results with the analysis of alignment and MEME. The phylogenetic analysis of these sequences indicated that the main character for cystatin genes might have been existent before the differentiation of Arabidopsis and rice. The cystatin domains among these proteins are highly conserved. The Arabidopsis and rice cystatin genes are expressed mainly in flower, leave, root, seed and callus, which is important for plant to defend the insects.(5) The high-affinity K+ (HAK) transporter gene family was the largest family in plant that functioned as potassium transporter and was important for various aspects of plant life. In present analysis, we identified 27 members of this family in rice in a genome-wide scale. The phylogenetic tree divided HAK transporter proteins into four distinct groups. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.The above results would lay a foundation for functional validation exercise aimed at understanding the role of the members of these gene families.