- Genome-wide identification and expression analysis of PUB genes in cotton. - Prediction analysis of subcellular localization showed that the PUBs in cotton were widely localized in cells, but primarily in the nucleus. - All PUB genes were involved in the response to abiotic stresses (including salt, drought, hot and cold) to varying degrees.. - In the pathway, E3 is critical for the. - Full list of author information is available at the end of the article. - identification of the specific substrate protein and can be found in the most species [3]. - In Arabidopsis , there are more than 1400 genes encoding functional components of the ubiquitination pathway, of which approximately 90%. - Previous studies reported 64 PUBs were identified in Arabidopsis [9], 77 in rice [10], 33 in Chlamydomonas reinhardtii [11], 101 in Chinese cabbage [12] and 125 in soybean [13], indicating that PUB genes are widely distributed in plants. - Approximately 50 cotton species were distributed in arid and semi- arid regions of the tropic and subtropics, which were pre- sumed to have originated from the same ancestor 50 to 100 million years ago [19]. - The number of PUB genes in tetraploid cottons was twice as high as that in diploid cottons, showing that PUB genes were relatively conser- vative. - The essential information about the gene name, chromosome locations, length of the open reading frame (ORF), type of protein domain, position of the U-box domain and subcellular localizations of these gene family members could be found in additional files (Additional file 2: Table S2, Additional file 3: Table S3, Additional file 4: Table S4 and Additional file 5: Table S5). - The length of the PUB protein sequence in the cotton ranged from 49 to 1492 AA, and the U-box domain contained approximately 75 amino acids. - However, the length of the U-box domain was almost identical except for a few PUBs. - Results of the subcellular localization analysis showed PUB pro- teins could be found throughout the cell, including nu- clear, cytoplasmic, chloroplast, plasma membrane, mitochondrial, and extracellular locations. - Structure and evolution analysis of PUBs in cotton A Gene structure diagram of the PUBs and an evolution tree were constructed (Additional file 6: Fig. - S2, Additional file 8: Fig. - Based on the evolutionary relationship, the PUB genes could be categorized into. - Among these subgroups, subgroup I was composed of the domains “U-box + ARM” and. - “U-box only”, and the remaining subgroups were com- posed of the other domains. - The exon number of PUB genes in cotton was greatly divergent, ranging from 1 to 25. - Among all the PUBs, approximately 1/3 of the PUBs contained only one exon. - Generally, the evolutionary rela- tionship is correlated with gene structure in some way, that is, exons with the more similarities in terms of the number and size of the exon, have a closer evolutionary relationship.. - hirsutum , the length of GhPUB1A is 47 Kb, much lar- ger than the other PUB genes, which may be correlated with the assembly and annotation of the cotton genome. - Chromosomal localization analysis of PUB genes in three cotton genomes. - The MapInspect software was used to analyze the localization of PUB genes on the chromosomes based on the position information. - These re- sults indicated that only a few genes were present on chromosomes 3, 4, and 12, and the chromosome 5 con- tained the highest number of PUB genes (11 PUBs). - In addition, PUB genes on chromosomes and 12 were preferentially enriched towards the end of the chromosome. - All of 96 PUB genes identified in G. - arboretum , chromosome 1 containing the most PUB genes (up to 14) and chromosome 3 con- taining the least PUB genes (only 2). - In addition, the length of chromosome 5 was approximately 6 Mb, how- ever 9 PUB genes were found on it, presenting the high- est distribution density. - hirsutum of the PUB genes were anchored onto chromosomes, as shown in Fig. - 2, among which 82 and 87 genes were found in the At- and Dt- subgenome, respectively. - The number of PUB genes on chromosome D07 was the most and chromosome D08 was the least compared with Table 1 Domain organizations of PUB proteins in cotton. - 1 Locations of PUB genes on chromosomes in G. - a, Locations of PUB genes on chromosomes in G. - b, Locations of PUB genes on chromosomes in G. - 2 Locations of PUB genes on chromosomes in in G. - hirsutum (Additional file 11:. - These results indicated that the PUB genes were equally distributed in At- and Dt- subgenomes but un- evenly localized on each chromosome, which may be correlated with the differentiation of these species.. - Fragment duplications in the genome region may result in the scattering of the gene family members. - In the study, BLAST2.2.31+ (ftp://ftp.ncbi.. - nlm.nih.gov/blast/executables /blast+/LATEST/) was used for BLASTN and BLASTP (value 10) screening of homolo- gous gene pairs from the cotton PUB genes identified. - The uneven distribution of PUB genes on the chromosome may be correlated with the gene duplication or partial fragment replication events during the long evolutionary history of the cotton genome. - Each time the replication event occurs, the entire genetic sequence of the cotton is doubled, and over time, these redundant genes are recombined or lost [23].. - Based on the multiple sequence alignment of the encoding sequences and the proteins in diploid cotton, 18 and 27 homologous gene-pairs were discovered with MCScanX [28] in G. - hirsutum , among which were located in the Dt-subgenome, and 191 homologous gene-pairs were found in both G. - hirsutum , among of which were lo- cated in the At-subgenome. - Expression pattern analysis of PUB genes in cotton Based on previous transcriptome data of the PUBs under different stresses (including salt, drought, hot and cold) in G. - hirsutum and 119 PUB genes were found with FPKM >. - Among all the PUB genes, approximately 21 non-expressed PUB genes were identi- fied in three tissues, and they may be associated with other specific regulation functions. - All the PUB genes were categorized into five subgroups (I, II, III, IV and V), and similar expression patterns were found among all PUB genes (Additional file 14: Fig. - In subgroup I, 18 PUB genes with pro- found expression differences were discovered. - in addition, other PUB genes in subgroup II- IV were found to have a consistent expression pattern under dif- ferent stresses. - However, 4 PUB genes ( GhPUB32A - GhPUB38D) in subgroup V showed a small expression difference under different stresses.. - To investigate the functions of the homolo- gous genes in cotton, qRT-PCR was used to investigate the expression difference in G. - Fifteen days after the VIGS infection, albino leaves of the positive control plants were observed, and all newly-emerged leaves were white in the later stage, while the others were normal with no albino leaves (Fig. - In this study, bio- informatics analysis was performed on allotetraploid cot- ton genomes AD 1 and AD2, and diploid cotton genomes A 2 and D 5 , and finally a total of 582 PUB genes were identified, including 185 genes in AD 1 genome, 208 in AD2 genome, 96 in A 2 genome, and 93 in D 5 genome, indicating that it was a relatively conserved family in. - raimondii has undergone at least two complete genome-wide replications [24, 30], resulting in an uneven distribution of the PUB genes on the chromo- somes, and over the time of the cotton evolution, some genes are reassembled or lost. - The results also showed that 19 of the 96 PUB genes in G. - arboreum were gener- ated through tandem repeats, which was one of the main reasons for the expansion of this gene family. - The classification of PUB protein differs from that of the other gene families - it depends not only on U-box homology but also on domains other than U-box do- mains [34]. - The evolutionary relationship of PUB genes between different cotton species is close, and the genetic structure in cotton is highly conserved. - During the process of cotton evolution, in addition to the U-box do- main, some other domains retained the basic functions of the family and enriched the diversity of PUB genes.. - Gene structure analysis showed that exon number of PUB genes varied greatly from 1 to 25, which might be due to the directional evolution in the function and structure of PUB genes during the long evolutionary his- tory. - All PUB genes could be divided into five subgroups (I-V) in each species according to the evolutionary rela- tionship, which was different with the classification of. - In the study, subgroup II and IV were found containing only 4 and 2 PUB genes, respectively. - In this study, 89% of PUB genes were differentially expressed in three tissues under salt, drought, cold and hot stresses, which also proved that PUB genes played important roles in. - Many studies have shown that PUB genes play an im- portant role in the process of stress responses in plants.. - In this study, two homologous PUB genes GhPUB85A and GhPUB45D were discovered with the same length of ORFs, type of protein, and subcellular lo- calizations. - In addition, these two PUB genes GhPUB85A and GhPUB45D were cloned and functionally evaluated. - The results in this study laid a foundation for the further study of PUB genes of in cotton in future.. - All CDS sequences identified and the genome sequence of the PUBs were used to analyze the gene structure with software GSDS2.0 [26]. - Multi-sequence alignment of the PUBs was carried out by ClustalX1.83, and Neighbor-Joining (NJ) method [45] was used to construct a phylogenetic tree in MEGA6.0 [27].. - GFF (general feature format) information of the cotton PUBs were obtained from the genome annotation files.. - The first strand was synthesized according to the instructions of the TransScript One-step gDNA Removal and cDNA Syn- thesis Supermix kit. - Additional file 1 Table S1. - Primers used in the manuscript.. - Additional file 2 Table S2. - Additional file 3 Table S3. - Additional file 4 Table S4. - Additional file 5 Table S5. - Additional file 6 Fig. - Additional file 7 Fig. - Additional file 8 Fig. - Additional file 9 Fig. - Additional file 10 Fig. - Additional file 12 Fig. - Additional file 13 Fig. - Additional file 14 Fig. - Additional file 15 Fig. - CC made contributions to the language polishing and modifications in the manuscript. - All authors have read and approved the final version of the manuscript.. - The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. - Genome-wide survey and expression analysis of the PUB family in Chinese cabbage (Brassica rapa ssp. - Genome sequence of the cultivated cotton Gossypium arboreum.. - Evolution of the protein repertoire. - Selection in the evolution of gene duplications. - Genome-wide identification and analysis of the stress-resistance function of the TPS (Trehalose- 6-phosphate synthase) gene family in cotton. - Identification and expression profile analysis of the protein kinase gene superfamily in maize development. - Extent of gene duplication in the genomes of Drosophila, nematode, and yeast
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