Abstract:

【Objective】 Expansins are a group of non-enzymatic proteins found in the plant cell wall, with important roles in plant growth, development, biotic and abiotic stress responses. To date, no systematic study on the molecular characterization, phylogeny and expression profiling of the upland cotton Expansin gene family has yet been conducted. In this study, a genome-wide identification, characterization and expression analysis of the Expansin gene family in upland cotton was performed. 【Method】 The members of the Expansin gene family in the upland cotton genome were identified by using the bioinformatics tools BLAST and HMMER, and were further analysed by using a combination of the bioinformatics softwares, such as ClustalW, MEGA, MCScanX, Prot Param, MEME, SignalP, Euk-mPLoc, Fancy Gene and DnaSP. The spatiotemporal expression patterns of the upland cotton Expansin gene family, and the differential expression of some Expansin homoeologs during the different stages of growth were determined by publicly available RNA-seq data. The expression patterns of some candidate Expansin genes were further validated by qRT-PCR. 【Result】 In the allotetraploid upland cotton, 72 expainsin-coding genes are identified, which is approximately twice as many as in the two diploid cotton species (Gossypium arboretum and G. raimondii), and these Expansin-coding genes are grouped into four subfamilies: 46 α-expansins (EXPAs), 8 β-expansins (EXPBs), 6 Expansin-like As (EXLAs), and 12 Expansin-like Bs (EXLBs). Except the two chromosomes GhA02 and GhD06, Expansin-coding genes are unevenly distributed across the other chromosomes ranging from 2 to 4, while the chromosomes GhA08 and GhD08 harbors 5 genes and 8 genes, respectively. Phylogenetic tree reveals that the members of the same subfamily are clustered together. In most cases, four Expansin members from the four (sub-)genomes of three cotton species (G. hirsutum, G. arboretum and G. raimondii) tends to cluster together within a given clade, for example, EXPA subfamily members Cotton_A_28454/Gh_A03G0885/Gh_D02G1269/Gorai.005G142200 which are located on collinear blocks are clustered into a clade. The computational prediction tool shows that all the Expansin proteins are predicted to be extracellular. The exon-intron structure analysis reveals that the upland cotton Expansin-coding genes typically consist of 3-5 exons interrupted by multiple introns, share an evolutionarily conserved exon-intron structure (consistent with the diversity of amino acid sequences), and have codon usage bias. RNA-seq data shows that different Expansin-coding genes are expressed in a stage- and tissue-specific manner during the developmental stages. For example, transcripts for GhEXPA19A and GhEXPA19D are highly abundant in the fire 10 days post anthesis (DPA) and 20 DPA when compared with other Expansin-coding genes. GhEXPA24D is highly expressed in few tissues, including cotyledons, new leaves, old leaves and bracts. Homoeologous genes exhibits different expression profiles, indicating the functional divergence and complementation. The qRT-PCR results are consistent with the RNA-seq data with the same trends for the expression of each Expansin-coding gene. For instance, GhEXLA3A and GhEXLA3D are highly expressed during the fiber elongation stage. GhEXPA19D and GhEXLA2D are highly expressed in the ovule at 3 DPA.【Conclusion】 The upland cotton genome contains 72 Expansin-coding genes which encode protein exhibiting the same structural diversity and evolutionary conservation as the coding DNA sequences of expansins, and which display diverse and dynamic expression patterns, implying functional conservation and divergence among the members of cotton Expansin genes.

Key words: G. hirsutum, Expansin, gene family, bioinformatics, gene expression