Genome-wide identi cation and expression analysis of PUB genes in cotton

Xuke Lu Chinese Academy of Agricultural Science Na Shu Hanzhong Agricultural Science Institute Delong Wang Chinese Academy of Agricultural Sciences Junjuan Wang Chinese Academy of Agricltural Sciences Xiugui Chen Chinese Academy of Agricultural Sciences Binglei Zhang Chinese Academy of Agricultural Sciences Shuai Wang Chinese Academy of Agricultural Sciences Lixue Guo Chinese Academy of Agricultural Sciences Chao Chen Institute of Cotton Research, Chinese Academy of Agricultural Sciences Wuwei Ye (  yew158@163.com ) Institute of Cotton Research, Chinese Academy of Agricultural Sciences https://orcid.org/0000-00034757-1057


Background
The ubiquitin-mediated ubiquitination pathway is the post-translational modi cation pathway of eukaryotic proteins. Studies have demonstrated that the pathway is involved in the cell cycles of higher plants [1], stress resistance [2], signal transduction [3], apoptosis [4], optical signal [5] and other physiological pathways. In the ubiquitin pathway, three steps are required for ubiquitin to act on the target protein. First, the ubiquitin-activating enzyme (E1) activates ubiquitin [1], and then the activated Ub molecules are delivered to the ubiquitin-binding enzyme (E2) [2]; nally, the Ub molecules are transferred to the target protein through ubiquitin ligase (E3). E3 is critical for the identi cation of the speci c substrate protein and is found in the most species [3]. In Arabidopsis, there are more than 1,400 genes encoding components of the ubiquitination pathway, of which approximately 90% encode ubiquitin ligase E3 [3,4]. According to its subunit composition and mechanism of action, the ubiquitin ligase E3 can be categorized into single subunit types (such as HECT, RING/U-box) [5] and multi-subunit type (such as SCF (skp1-cullin-F-box), APC (anaphase-promoting complex) [6], VBC (VHL-Elongin B-Elongin C), etc.) [7,8].
PUBs have been reported in many model crops, including Arabidopsis, rice, Chlamydomonas reinhardtii, Chinese cabbage, and soybean. Previous studies identi ed 64 PUBs 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 widely distributed and play important roles in plants. Many studies have shown that PUB proteins are involved in abiotic stress responses. Cho et al. [14] obtained U-box E3 protein (CaPUB1) from drought-treated peppers. In Arabidopsis, the heterologous overexpression of CaPUB1 attenuated the tolerance of plants to drought stress, and two homologous proteins AtPUB22 and AtPUB23, in Arabidopsis are also involved in the drought stress response of the plants [15]. Furthermore, studies also showed that AtPUB22 and AtPUB23 can negatively modulate the drought stress response of plants by synergistic ubiquitination of RPN12a [15]. Liu et al. identi ed a U-box E3 protein AtPUB19 induced by drought, high salt levels, cold and ABA. Mutated atpub19 in plants promoted stomatal closure, thereby enhancing the tolerance to drought; AtPUB19-overexpressing plants were not sensitive to ABA but were sensitive to drought. In addition, there are signi cant changes in the expression levels of ABA and stressrelated genes in atpub19 mutant and AtPUB19-overexpressing plants, indicating that AtPUB19 may regulate the drought stress by negatively modulating the ABA signaling pathway [16]. PUB proteins, involved in the process of drought response, were also identi ed in rice. Previous studies showed that the drought resistance of OsPUB15-overexpressing plants was signi cantly enhanced, and OsPUB15 could be induced by hydrogen peroxide, drought and high salt levels, indicating that OsPUB15 may positively regulate the response of plants to drought stress by attenuating intracellular oxidative stress [17].
Cotton is the most important ber crop and the model crop for research into polyploidy, evolution, cell wall development, and cellulose synthesis [18]. Approximately 50 species were distributed in arid and semi-arid regions of the tropic and subtropics, which were presumed to have originated from the same ancestor 50 to 100 million years ago [19]. The current cultivars are diploid G. arboreum and G. herbaceum, and tetraploid G. hirsutum and G. barbadense. The tetraploid cottons originated from the hybridization of an African ancestral species with A genome and an American ancestor species with D genome one to two million years ago [18] . Recently the sequencing work of diploid cottons (G. raimondii (D5) [20,21], G. arboreum (A2) [22] and allotetraploid cottons (G. hirsutumTM-1 (AD1) [23,24], G. barbadense acc.3-79 (AD2) [25] were completed, providing references for the study of gene function and evolution at the whole genome level. Based on the cotton genome sequences, the PUB gene family members of the PUB gene were identi ed and the structure and distribution characteristics of PUB genes were analyzed, and their evolution analysis was analyzed in whole-genome replication and cotton allotetraploid formation studies.

Results
Identi cation of PUB gene family members in the whole genome of cotton The hidden Markov model (HMM) of the U-box domain (PF04564) was downloaded from the Pfam30.0 database, and used as a query to identify the candidate PUB members in four cotton genomic database using HMMER3.0. SMART. In addition, Pfam30.0 was also used for further identi cation to con rm every PUB members containing U-box domain. Finally, 93, 96, 185, and 208 PUBs were identi ed from the four sequenced cotton species, G. raimondii (D 5 ), G. arboreum (A 2 ), G. hirsutum acc. TM-1 (AD 1 ), and G. barbadense (AD2), respectively, and these PUBs were named GrPUB1-93, GaPUB1-96, GhPUB1A-89A/1D-91D/181-185 and GbPUB1A-98A/1D-98D/197-208 according to their location on the chromosome. The number of PUB genes in tetraploid cottons was twice as high as that of diploid cottons, showing that PUB genes are relatively conservative. The 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 les (Additional le 2: Table S2, Additional le 3: Table S3, Additional le 4: Table S4 and Additional le 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; for example, GaPUB39 and GhPUB40D had only 32 and 50 amino acids, respectively. Results from the subcellular localization analysis showed PUB proteins distributed throughout the cell, including nuclear, cytoplasmic, chloroplast, plasma membrane, mitochondrial, and extracellular locations. However, most PUB proteins were located inside the nucleus. Twenty different domains were found among all the cotton PUBs (Table 1), and the primary mode was "U-box+ARM/HEAT". Different domain modes may be associated with different functions of cotton PUBs.

Analysis of PUBs gene structure and evolution in cotton
A Gene structure diagram of the PUBs and an evolution tree were constructed (Additional le 6: Figure S1, Additional le 7: Figure S2, Additional le 8: Figure S3 and Additional le 9: Figure S4). Based on the evolutionary relationship, the PUB genes could be categorized into ve subgroups (I-V). Among these subgroups, the subgroup I was composed of the domains "U-box + ARM" and "U-box only", and the remaining subgroups were composed of the other domains. The exon number of PUB genes in cotton is greatly divergent, ranging from 1 to 25. Among all the PUBs, approximately 1/3 of the PUBs contained only one exon. Generally, the evolutionary relationship 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 the evolutionary relationship. In G. hirsutum, the length of GhPUB1A is 47 Kb, much larger than the other PUB genes, which may be correlated with the assembly and annotation of the cotton genome. Members in each subgroup of G. barbadense (AD2) was much different than those in G. raimondii (D 5 ), G. arboreum (A 2 ) and G. hirsutum (AD 1 ), and this difference may be correlated with the different origins of these species. Therefore, the evolution of the PUBs in G. raimondii (D 5 ), G. arboreum (A 2 ) and G. hirsutum (AD 1 ) was analyzed, and the results also indicated -subgroups were found (Additional le 10: Figure S5), and this was similar with the evolution of single PUB gene, suggesting the PUB members were highly conservative. Furthermore, closer evolution relationships of GhPUB1A-89A with GaPUB1-96 and GhPUB1D-91D with GaPUB1-93 were found through the evolutionary analysis.

Location of PUB genes in three cotton genomes
The MapInspect software was used to generate the distribution map of PUB genes on the chromosomes based on the position information. Among 93 genes in G. raimondii, 91 were located unevenly on the chromosome and the others were found on scaffolds ( Figure 1A). These results indicated that only a few genes were present on chromosomes 3, 4, and 12, and the chromosome 5 contained the highest number of PUB genes (11 PUBs). In addition, PUB genes on chromosomes 4, 6, 7, 11 and 12 were preferentially enriched towards one end of the chromosome. All of 96 PUB genes identi ed in G. arboreum were located on different chromosomes ( Figure 1B). The results showed uneven distribution of PUBs on each chromosome in G. arboretum with chromosome 1 being the most enriched with PUB genes (up to 14) and chromosome 3 being enriched the least with PUB genes (only 2). In addition, the length of chromosome 5 was approximately 6 Mb, but 9 PUB genes were found on it, showing the highest distribution density. In G. hirsutum, 91.4% (169/185) of the PUB genes were anchored onto chromosomes, among which 82 and 87 genes were found in the A-and D-subgenome, respectively ( Figure 2). The number of PUB genes on chromosome 7 was the most and chromosome 8 was the least compared with other chromosomes in both At-and Dt-subgenomes of G. hirsutum, showing that PUBs on these two chromosomes were relatively conserved and signi cant for cotton growth. The situation for G. barbadense was different with that of G. hirsutum (Additional le 11: Figure S6). The results showed that the PUB gene were equally distributed in At-and Dt-subgenomes and unevenly located on each chromosome, which may be correlated with the differentiation of these wo species.
Gene duplication analysis Myr years ago [18], and allotetraploid G. hirsutum was generated from the hybridization of diploid cottons and the number of chromosomes were doubled 1-2Myr years ago. 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 homologous gene pairs from the cotton PUB genes identi ed. The uneven distribution of genes on the chromosome may be the result of gene duplication or partial fragment replication during the long evolutionary history of the cotton genome. Each time the replication occurs, the entire genetic sequence of the cotton is doubled, and over time, these redundant genes are recombined or lost [23]. Previous studies have shown that gene duplication and post-segregation phenomena are two major driving forces of evolution [27]. 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. raimondii (D 5 ) (Additional le 12: Figure S7A) and G. arboreum (A 2 ) (Additional le 12: Figure S7B), respectively. Among these homologous gene-pairs, 15 segmental duplications and 3 tandem duplications were found in G. raimondii, and 25 segmental duplications and 2 tandem duplications were found in G. arboreum. The relationship between these two diploid cottons and G. hirsutum was analyzed (Additional le 13: Figure S8). Totally 197 homologous gene-pairs were found between G. raimondii and G. hirsutum, among which 58.89% (116/197) were located in the Dtsubgenome, and 191 homologous gene-pairs were found in both G. arboreum and G. hirsutum, among of which 55.50%(106/191) were located in the At-subgenome. All these results showed that more than one half of homologous genes in G. hirsutum were derived from the corresponding diploid cotton genomes. Furthermore, approximately 41.11% -44.50% of these homologous genes were originated from other diploid genomes.

Expression pattern analysis of PUB genes in cotton
Based on previous transcriptome data of the PUBs under different stresses (salt, drought, hot and cold) in G. hirsutum, 117, 148 and 119 PUB genes were found with FPKM >1 in roots, stems and leaves, respectively, thus displaying tissue speci city. Among all the PUB genes, approximately 21 non-expressed PUB genes were identi ed in three tissues, and they may be associated with other speci c regulation functions. Interestingly, it was found that 3 PUB genes (including GhPUB58D, GhPUB55A and GhPUB67D) were always highly expressed in three tissues under salt, drought, cold and hot stresses. All the PUB genes were categorized into ve subgroups (I, II, III, IV and V), and similar expression patterns were found among all PUB genes (Additional le 14: Figure S9 and Additional le 15: Figure S10). In subgroup I, 18 PUB genes with profound expression differences were discovered; in addition, other PUB genes in subgroup II-IV were found to have a consistent expression pattern under different stresses. However, 4 PUB genes (GhPUB32A -GhPUB38D) in subgroup V showed a small expression difference under different stresses.
Cloning and functional analysis of GhPUB85A and GhPUB45D The evolutional relationship in Additional gure S5 showed GhPUB68A, GhPUB85A, GhPUB45D and GhPUB69D were belonged to subgroup III, indicating that their close relationship with each other. The transcriptome data showed that GhPUB85A and GhPUB45D were highly expressed whereas GhPUB68A and GhPUB69D were negligibly expressed. To investigate the functions of the homologous genes in cotton, qRT-PCR was used to investigate the expression difference in G. hirsutum TM-1. Drought, salt and cold treatments were applied and the results were present in Figure 3. High expression of GhPUB85A and GhPUB45D under three stresses suggested that they were actively expressed to respond the abiotic stresses, but GhPUB68A and GhPUB69D were not highly expressed, which was in line with previously reported transcriptome data. Interestingly, we found that GhPUB85A and GhPUB45D were highly expressed at 6h under drought stress, while the expressions at 12h were the highest under salt and cold stress, indicating that GhPUB85A and GhPUB45D responded to drought stress faster than they did to salt and cold stresses. However, the expression values of GhPUB85A and GhPUB45D were signi cantly different under the same stress conditions, showing their different contributions in responding to abiotic stresses.
In addition, GhPUB85A and GhPUB45D were cloned using cDNA from G. hirsutum TM-1, and ligated to pEASY-Blunt Cloning Vector for sequencing to verify whether the vector was correctly ligated. The sequencing and enzyme digestion results showed that the recombined vectors were correctly constructed.
Red uorescence vectors pBI121-GhPUB85A:RFP and pBI121-GhPUB45D:RFP were constructed to research their subcellular localizations (Figure 4), and the results showed that these two genes were located at the cytomembrane, which were consistent with our prediction in Additional table S2. In addition, two VIGS vectors pYL156:GhPUB85A and pYL156:GhPUB45D were constructed using In-Fusion technology to study their functions under different stresses. 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 (Figure 5a). We investigated the expression quantity in the control plants (CK), and pYL156-, pYL156:GhPUB85A-and pYL156:GhPUB45D-infected plants under different stresses. The expression levels of two genes decreased signi cantly after the VIGS infection under different treatments showed their positive functions in responding to multiple stresses and the success of VIGS infection (Figure 5b-d), indicating the VIGS infection technology was an effective way to study the gene functions in cotton.

Discussion
The PUB gene family has been identi ed and analyzed in a number of plants [10,[12][13][14]. In this study, bioinformatics analysis was performed on allotetraploid cotton genomes AD 1 and AD2, and diploid cotton genomes A 2 and D 5 , and nally a total of 582 PUB genes were identi ed, 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 is a relatively conserved family in terms of cotton genome evolution. Whole-genome replication analysis revealed that the ancestors of G. arboreum. and G. raimondii had undergone a cotton-speci c genome-wide replication event that occurred about 1.6 million years ago after the differentiation from cocoa about 33 million years ago [29]. Recent studies have shown that G. raimondii has undergone at least two complete genome-wide replications [24,30], resulting in an uneven distribution of the PUB genes on the chromosomes, 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 generated through tandem repeats, which was one of the main reasons for the expansion of this gene family. Gene duplication event is a common phenomenon in plants, including multiple forms, such as tandem duplication, segmental duplication, and whole-genome duplication [31,32]. Some of duplicated genes could be retained in its descendants, which could provide original genetic resource for adaptive evolution of plants [33]. In this research, gene duplication event was commonly found, totally 31 and 27 gene pairs were discovered in D5 and A2 genome, respectively. In G. hirsutum, more gene pairs were found than the sum of A2 and D5, which might be associated with the higher resistance and wider adaptability of G. hirsutum.
The classi cation of PUB protein differs from that of the other gene families -it depends not only on Ubox homology but also on domains other than U-box domains [34]. The evolutionary relationship of PUB genes between different cotton species is close, and the genetic structure in cotton is highly conserved.
During the evolution of cotton, in addition to the U-box domain, 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 history. All PUB genes could be divided into ve subgroups (I-V) in each species according to the evolutioanary relationship, which was different with the classi cation of U-box containing genes in C.reinhardtii [11]. In the study, subgroup II and IV were found containing only 4 and 2 PUB genes, respectively.These ndings suggested that the gene structures and evolutionary relationship of PUB family members were signi cantly different between different species. Previous studies have demonstrated that plant U-box containing genes are widely involved in stress responses, disease resistance and nutrient defect responses in plants [35][36][37]. 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 an important roles in abiotic responses. Twenty two PUB genes, including 18 genes from GhPUB8A to GhPUB51D in subgroup I and 4 genes from GhPUB32A to GhPUB38D in subgroup V, showed an signi cant expression difference under different abiotic stresses, suggesting that they evolved toward speci c functions in the long history. This perspective was consistent with previous documents [38,39].
Many studies have shown that PUB genes play an important role in the process of stress tolerance in plants. For example, the overexpression of AtCHIP gene in Arabidopsis results in its sensitivity to high temperature and low temperature [40], while the AtPUB18, AtPUB19, AtPUB22 and AtPUB23 respond positively to drought [16, 30,41]. In this study, two homologous PUB genes GhPUB85A and GhPUB45D were discovered with the same length of ORFs, type of protein, and subcellular localizations. The only difference was their locations on chromosomes, GhPUB85A on chromosome 7 in At-subgenome while GhPUB45D on chromosome 7 in Dt-subgenome, which was not reported before. In addition, these two PUB genes GhPUB85A and GhPUB45D were cloned and functionally evaluated. Signi cant expression differences revealed that the functions of these homologous genes were similar in response to abiotic stresses, but their contributions differed from each other. Both of GhPUB85A and GhPUB45D contained the protein domain TPR, which was the same with AtCHIP, so we speculated that the functions of these two genes might be similar with AtCHIP [42], at least in response to cold stress which had been veri ed in the study. The results from this study laid a foundation for the further study of PUB genes of in cotton in future.

Conclusions
The identi cation and expression analysis of PUB family members in cotton in this manuscript provided insights into response mechanism to abiotic stresses. Although the PUBs were highly conserved throughout the evolutionary history of cotton, signi cant differences were found between each other in gene structure. In addition, two homologous genes GhPUB85A and GhPUB45D were cloned and functionally identi ed. Expression pattern analysis showed they both responded to abiotic stresses positively, but their contributions were different. Therefore all these results were of great signi cance for the future research of molecular mechanism in responding to abiotic stresses.

Cotton and Arabidopsis thaliana
Upland cotton variety ZhongS9612 was selected and has been preserved by the Cotton Adversity Research Laboratory at the Chinese Academy of Agricultural Sciences (CAAS) for many years. Before planting into sand, the seeds were surface-sterilized with 0.1% HgCl 2 and placed in a sterile dish with moist lter paper to accelerate germination. Uniform seedlings were chosen and transplanted into sand pots (10 plants in each pot) in a greenhouse (14 h/day at 30°C and 10 h/night at 24°C) at the Institute of Cotton Research of CAAS. The cotton seedlings were treated with 200 ml of 30% PEG 6000 and 200 ml 400 mM of NaCl solution, which could achieve completely consistent stress environments in each pot. For the cold treatment, cotton seedlings were transferred into a 5°C refrigerator with clear glass, but the light condition was not changed. For the planting of Nicotiana benthamiana, we rst placed the seeds on MS medium at 22°C in a growth chamber with a 16-h light cycle for the germination. After the emergence, the tobacco seedlings were moved to nutrition-enriched soil for growth under the same condition for about 45 days. After the treatment, plant leaves were harvested and frozen with liquid nitrogen for use. For the agrobacterium-mediated transformation, we referred the method used by Lu et al [43].

Exon-intron structure, phylogenetic and conserved domain analysis
The identi ed CDS and the genome sequence of the cotton PUBs were used to analyze the PUBs structure by GSDS2.0 [26]. The full-length sequences of PUB proteins were used to construct a phylogenetic tree. Multi-sequence alignment of the PUBs was carried out by ClustalX1.83, and Neighbor-Joining (NJ) method [47] was used to construct a phylogenetic tree in MEGA6.0 [27]. The online software SMART, PROSITE [29] was used to analyze the conserved domains of each protein.
Physical location of PUBs in cotton GFF (general feature format) les of the cotton PUBs were obtained from the genome annotation les, and the information on PUBs of chromosome positions was predicted from it. The distribution of cotton PUBs on the chromosome was generated with MapInspect (http://mapinspect.software. informer. com/). Gene duplication and micro-synteny analysis of G. arboreum, G. raimondii and G. hirsutum L.
Homologous gene pairs were identi ed according to multiple sequence alignment results and the standard was described in previous studies [48,49]. The collinearity of homologous genes was visualized with program Circos-0.69 [50] based on the homology between each species and their positions on the genome.

Gene cloning and the construction of vectors
The rst strand was synthesized according to the instructions of the TransScript One-step gDNA Removal and cDNA Synthesis Supermix kit. Two homologous genes GhPUB85A and GhPUB45D were cloned using In-fusion technology. Primers were designed using the website and sequences were present in Additional le 1: Table S1. The PCR ampli cation products were veri ed using 1.5% agarose gel electrophoresis. PCR ampli cation products were puri ed using the MiniBEST Agarose Gel DNA Extraction Kit from Takara Corporation. Finally the concentration of targets was measured, and stored at -20°C for use.
Puri ed targets were linked to the pEASY-Blunt Cloning Vector, and then transformed into E. coli according to the instructions of pEASY-Blunt Cloning Kit. The transformation was conducted with heatshock method, which was a classically effective approach. First a mixture of vectors and products was prepared with a mole ratio of 1:7 before the reaction in 200 μl centrifuge tube at 25°C for 5 min. Second blend the linked products and E. coli DH5α cells when E. coli DH5α cells began to dissolve, then the reaction was performed on the ice for 30 min. Thirdly transfer the centrifuge tubes to 42°C water for 90 s to end the reaction, at last placed the centrifuge tubes on the ice for use. Positive clones were selected and inoculated into LB liquid medium containing Kana (50 mg•L -1 ) for about 6h under the conditions of 200 rpm and 37°C. Then the positive clones were veri ed using PCR ampli cation with primers. Finally cloning vectors GhPUB85A-t and GhPUB45D-t were obtained. PCR reaction system used in the research was below: 5 × PrimerSTAR GXL Buffer, 10.0 μl; dNTP Mixture (2.5 mM each), 4.0 μl; F-primer (10 μM), 1.5 μl; R-primer (10 μM), 1.5 μl; cDNA, 100 ng; 5×PrimerSTAR GXL DNA Polymerase, 100 ng, 5×PrimerSTAR GXL DNA Polymerase, 2.0 μl; add ddH 2 O to 50.0 μl. PCR procedure of gene ampli cation used in the research was: 98 °C, 10s; 55 °C, 15s; 68 °C, 9s; 4°C, forever, 35cycles. Real-time PCR method was used to measure the relative expression of two genes. Primers of two genes were listed in Additional le 1: Table  S1. GhHistone3 gene (AF02471) was used as the reference gene.

VIGS analysis of GhPUB85A and GhPUB45D
Based on the vector sequence, target gene sequence and enzyme cutting sites, In-fusion primers were designed at the website (http://bioinfo.clontech.com/infusion). Primer sequences of InGhPUB85A-V and InGhPUB45D-V were previously shown. Primer sequences of GhPUBs-RFP and InGhPUBs were listed in Additional le 1: Table S1. Finally the silencing vectors pYL156:GhPUB85A and pYL156:GhPUB45D, and RFP vectors pBI121-GhPUB85A:RFP and pBI121-GhPUB45D:RFP, and the Plant overexpression vectors pBI121:GhPUB85A and pBI121:GhPUB45D were totally constructed. Vectors were transformed into cotton and Arabidopsis thaliana with agrobacterium mediated genetic transformation method. Locations of PUB genes on chromosomes in in G. raimondii and G. arboreum Ga01-Ga13 and Gr01-Gr13 represent the chromosome 1 to chromosome 13 in G. arboretum and G. raimondii, respectively.

Figure 2
Locations of PUB genes on chromosomes in in G. hirsutum Replace the chromosome 1 to chromosome 13 of A subgenome of G. hirsutum with GhA01-GhA13, Replace the chromosome 1 to chromosome 13 of D subgenome of G. hirsutum with GhD01-GhD13.

Figure 3
Page 20/22 Expression patterns of GhPUB68A GhPUB85A GhPUB45D and GhPUB69D during the drought, salt or low temperature stress a, b and c represent drought, salt and low-temperature treatment, respectively.
Different letters from A to S indicate signi cance expression of different genes during different stresses (p < 0.01).

Figure 4
Subcellular localization of GhPUB85A and GhPUB45D