Identification and Expression Analysis of Tubulin Gene Family in Upland Cotton

doi:10.21203/rs.3.rs-125041/v1

Download PDF

Research

Identification and Expression Analysis of Tubulin Gene Family in Upland Cotton

https://doi.org/10.21203/rs.3.rs-125041/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 13 Jul, 2021

Read the published version in Journal of Cotton Research →

You are reading this latest preprint version

Background: Upland cotton (Gossypium hirsutum) is one of the important cash crops. Therefore, improving fiber quality of upland cotton, is one of the current important research directions. Cotton fibers are single-celled extensions of the seed epidermis, which is important pattern material of research cytoskeleton. Tubulin genes act an important role in the synthesis of the microtubules (MT), which is core element of the cytoskeleton. Some tubulin genes in cotton ovule and fiber development are were reported, while the systematic study of its family genes has not yet in cotton. Therefore, identification and expression analyses of G. hirsutum tubulin genes provide pivotal targets for molecular manipulation in cotton breeding.

Result: In this study, we investigated all tubulin genes from seven varies plant species, and identified 98 tubulin genes in G. hirsutum. Phylogenetic analysis showed that tubulin family genes were classified into three subfamilies. The protein motifs and gene structure of α, β-tubulin genes are more conservative comparing with γ-tubulin genes. Most tubulin genes are located at the proximate ends of the chromosomes. Spatiotemporal expression pattern by transcriptome and qRT-PCR analysis revealed that 12 α-tubulin and 7 β-tubulin genes are specific expression in different stages of fiber development. However, Gh.A03G027200, Gh.D03G169300 and Gh.A11G258900 had clear express pattern at distinct stages of ﬁber development in J02508 and ZRI015.

Conclusion: In this study, evolutionary analysis showed that the tubulin genes were divided into 3 clades. The genetic structures and molecular functions were highly conserved in different plants. Three candidate genes of Gh.A03G027200, Gh.D03G169300 and Gh.A11G258900 may play key roles during length and strength in fiber development with the expression analysis at different organizations.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Upland cotton

Fiber quality

Cytoskeleton

Microtubules (MT)

Tubulin

The production of upland cotton (G. hirsutum) accounts for great proportion of the global total cotton production in the textile industry. The advantage of high production of upland cotton is difficult to cover the flaw of poor fiber quality (Su et al. 2018). Therefore, improving the fiber quality of upland cotton is one of the current important research directions. Fiber length and strength are two of the important indicators in fiber quality (Gao et al. 2019). And, the process of cotton fiber development in extreme materials should be extensively explored. The development of cotton fibers can be delineated into four distinct overlapping stages: fiber initiation, elongation, secondary wall synthesis and maturation, which is a highly revised, basic biological process (Gao et al. 2007; Wang et al. 2017).Many studies have shown that the actin and tubulin genes played an important role in the synthesis of the cytoskeleton and cotton fiber elongation (Pydiura et al. 2019; Li et al. 2005). Microtubules (MTs) are core element of the cytoskeleton and act a pivotal function in cellular migration, mitosis, mechanical stress, cell polarity, intracellular transport, cell division, and cell morphogenesis (Nieuwenhuis et al. 2019). The tubulins are the extremely plentiful proteins (Findeisen et al. 2014). Most studies revealed the existence of genes from alpha, beta, and gamma tubulin families that were usually reference genes by qRT-PCR in all eukaryotes, which are highly conserved in structure and function (Findeisen et al. 2014; Zhao et al. 2014; Jayaswal et al. 2019; Ray et al. 2014). Most tubulin genes have difference in configuration merely due to few amino acid substitutions, which have two conserved domain Tubulin and Tubulin_C (Mubeen et al. 2016). In Arabidopsis thaliana, tubulin family included six α-tubulin and nine β-tubulin. All α- and β-tubulin tie GTP, which functions similarly to the ATP band to actin to adjust polymerization. It is indicated that α-tubulin forms hydrogen bonds with the GTPase domain of β-tubulin (Šutković et al. 2014). Generally, plant α-tubulin tyrosine nitration performs functions as posttranslational modification, the NO signal transduction with the participation of microtubules and one of the feature of the enhanced microtubule dynamics (Blume et al. 2013). Superfamilies or subfamilies of Tubulin genes were already reported in some species, such flax (Linum usitatissimum) (Gavazzi et al. 2017; Pydiura et al. 2019), fungal (Zhao et al. 2014), Salix arbutifolia (Rao et al. 2016).

Microtubules are chiefly built up heterodimers of α-tubulin and β-tubulin fastened, while γ-tubulin may only mediate microtubule nucleation, which are closely related to cellulose and microfiber deposition and play a key role in the development of secondary cell walls in all plants. Reduction of α-tubulin can disrupt the microtubule structure and further resulted deviant cell expansion (Rao et al. 2016). The tubulin proteins can easily be purified from cell suspension cultures of tobacco and Arabidopsis (Hotta et al. 2016), which may be intuitively study its structure and function for laying a good foundation in vitro research. Individual members of the tubulin family show organizational differences in various organizations (Rao et al. 2016). The γ-tubulin gene GCP6 is key for spindle morphogenesis but not necessary for microtubule reorganization in Arabidopsis (Miao et al. 2019). TUA1 in transgenic poplars had high expression in leaves, but abnormally low in xylem, indicating that high expression levels of tubulin protein were not tolerated in wood-forming tissues. As in leaves, the relative transcriptional abundance of the TUB is lower than the relative transcriptional abundance of the TUA in transgenic poplars. Interfering with tubulin proteins also affects the behavior of guard cells, delaying stomatal closure due to drought and light-induced leaf stomatal opening, demonstrating tubulin involve in non-cellulosic polysaccharide assembly during cell wall biogenesis (Swamy et al. 2015). The expression of α-tubulin protein may inhibit ear elongation during water deficiency. Defects in function and genotype of the α-tubulin genes are closely related to phenotype. In Monocotyledon plant such as rice, homozygous mutations in the α-tubulin genes cause extreme dwarfing and weak stems (Sheoran et al. 2014). In heterotetraploid tef (Eragrostis tef), there is only one α-tubulin gene mutation, and the second complete α-tubulin gene is overexpressed, which conferred a favorable semi-dwarf and lodging tolerance phenotype in agriculture (Jöst et al. 2015). The short seed phenotype of the Srs5 mutation (an α-tubulin) in rice has been shown to be due to reduced cell length (Segami et al. 2012). Gzyl et al. (2015) obtained results reveal the great impact of Cd on genes expression levels and subsequent post-translational processing of tubulin, which may be associated to the impairment of MT cytoskeleton in root cells.

The upland cotton β-tubulin genes were cloned and analyzed the expression levels from developing upland cotton ovules (He et al. 2008). However, the tubulin genes superfamily are less understood in cotton. In our study, we analyzed phylogeny relationships, gene structures, chromosomal locations, expression patterns. Further, the information about the tubulin gene family may be useful for further studies.

Plant Material and Nucleic Acid Extraction

Two upland cotton varieties (J02508 and ZRI015) with great differences to cotton ﬁber length and strong, were grown in Anyang Farm, China. Cotton ﬁbers at 0, 1, 3, 5, 10, 15, 20, 25 days post anthesis (DPA), root, stem, and leaves were collected for extraction of RNA. Each period for different organizations had three biological replicates. All samples were kept in liquid nitrogen for transportation and stored at -80°C. Total RNA was extracted using the RNAprep Pure Plant Plus Kit (TIANGEN). Electrophoresis and spectrophotometric detection were adopted to detect the quality and quantity of the nucleic acids. Then, using the RNA as the template, every sample contained 1 µg RNA. Following this, the cDNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Takara Biotechnology Co, Ltd., Dalian, China).

Sequence retrieval and phylogenetic analysis

The G. hirsutum genome sequences were obtained from the CottonGen website (https://www.cottongen.org). To identify tubulin genes in the genome of cotton, Arabidopsis, flax and rice plant tubulin gene family on wide-genome sequences which were respectively made discovery and identified, were downloaded from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) (Swarbreck et al. 2008; Pydiura et al. 2019). BLASTP with default parameters was used to identify further the tubulin proteins in cotton with Arabidopsis and flax tubulin sequences as the queries based on homology search. The selected cotton tubulin proteins were used for further identification by searching the cotton database again. These identified sequences domain were performed to make clear whether all protein sequences contain tubulin conserved domian using InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan). Subsequently, HMMER software with default parameters and conserved tubulin domains were used to search for above sequences again. Using same method, we obtained other three cotton predicted tubulin genes, including G. barbadense, G. arboreum and G. raimondii. Multiple sequence alignment of all identified tubulin proteins was performed using ClustalX with default parameters (Thompson et al. 1997). Phylogenetic trees were constructed using bootstrap Neighbor-Joining algorithm with bootstrap analysis of 1,000 replicates in MEGA7 v7.0.26 (Kumar et al. 2016).

Gene structure, chromosomal mapping and collinearity analysis

The Gene Structure Display Server Program (http://gsds.cbi.pku.edu.cn/) was employed to derive exon-intron structure representations based on basic coding information as described above. All motifs were identified by MEME software (http://meme-suite.org/) at tubulin protein in G. hirsutum genome. Relational gene chromosomal position information for all G. hirsutum tubulin sequences was obtained from annotation files downloaded from the CottonGen website and visualized by tool CIRCOS to illustrate the chromosomal distribution. We used Circos to draw the distribution (Zou et al. 2013). The collinearity pairs of tubulin family were mapped adopting Circos software (Krzywinski et al. 2009).

Gene Expression Analysis

The RNA-seq data of five distinct tissues from two upland cotton varieties J02508 and ZRI015 were already upload to the NCBI Gene Expression repository. The reference genome sequences for G. hirsutum were downloaded (Yang et al. 2019). The FPKM values of tubulin genes were calculated using the Cuﬄinks program. The gene expression data (FPKM) were divided by the mean of all the values, then were normalized with the log₂ (FPKM+1) method to calculate the expression levels. Gene expression patterns between diﬀerent ﬁber developments were visualized with heat maps using the TBtools software package (Chen et al. 2020).

The gene-speciﬁc primers used for qRT-PCR were designed with a primer database (http://biodb.swu.edu.cn/qprimerdb). All the primers were listed in table 1. The authors used the GhUBQ (GhA10G005800) gene as an internal control, and a total volume of 20 µL that contained 0.4 µL of each primer (10 µM), 2 µL cDNA, 10 µL SYBR Premix Ex Taq(2×), and ddH₂O to make up the volume used to perform qRT-PCR with three biological and technological replicates on ABI 7500 fast real-time PCR system using SYBR Premix Ex Taq (Takara). The gene relative expression levels were calculated using the 2⁻^△△^Ct method.

Phylogenetic analysis of the tubulin protein family

To investigate the evolutionary relationships of tubulin genes, the tubulin protein sequences from O. sativa, Arabidopsis, L. usitatissimum, G. arboreum, G. barbadense, G. raimondii, and G. hirsutum were used to generate an unrooted phylogenetic tree (Fig.1). In those species, the tubulin proteins were mainly classified into 3 bunches e.g. α, β and γ-tubulins, which revealed similar results to previous studies in different plants (Jayaswal et al. 2019). Most of the orthologous genes between the allotetraploid and the corresponding diploids were clustered into the same clade based on phylogenetic analysis with tubulin protein sequences, which indicates that before the separation of monocots and dicotyledons, the genes had been differentiated into 3 different subfamilies. In each bunch, most tubulin proteins from four cotton species were relatively farther than tubulin proteins from O. sativa, Arabidopsis, L. usitatissimum, but a few had special case including some genes in O. sativa, which closely related to cotton species. γ tubulins are abundant gathering together from O. sativa, which may play a vital role in development of rice, but which are relatively scarce from Arabidopsis, only AT.5G05620 and AT.3G61650. Interestingly, the α, β-tubulins subfamilies are extended, but γ-tubulin subfamily is relatively small in cotton. The above results show that tubulin genes are lost or expanded during evolution, according to the needs of plant growth.

Gene structure conserved motifs analysis

In this study, we analyzed the conserved motif and structural characterizations to further investigate the phylogenetic relationships among diﬀerent members of the tubulin family of G. hirsutum (Fig.2). Members of α, β-tubulin genes are quite conservative, yet γ-tubulin genes are not retained in protein motifs and gene structure analysis. There was a considerable distinction in the exon-intron structure in different branches, but similar in the exon-intron structure in same branches. Most of the genes had four introns in α-tubulin subfamily, and had three introns in β-tubulin subfamily, yet had five to eleven introns in γ-tubulin subfamily. The MEME program was used to identify 10 conserved motifs. There are almost all conserved sites in α, β-tubulin subfamilies. However, the genes of Gh.D05G257500, Gh.D12G088700 and Gh.D04G181300 may lose one or two of motifs 4/5/8 in α, β-tubulin subfamily. Most γ-tubulin genes are poorly conserved in compared to member of α, β-tubulin genes. Strangely, the Gh.A11G102000, Gh.D11G102600 and Gh.A12G127300 contain ten conserved motifs in γ-tubulin subfamily. The above results show that most members from the same subfamily have similar motif features, and exon-intron structure, supporting the close evolutionary relationships.

As the (table 2/Fig. S1), the results showed that the most α, β-tubulin proteins have conserved domains with Tubulin and Tubulin_C; pIs of the most tubulin proteins are weakly acidic, however, when the proteins loss the Tubulin_C, the pIs of proteins are alkaline. Conserved site analysis of tubulin gene family were found that some amino acid are very conserved (Fig. S2), The BaCelLo (Pierleoni et al. 2006), EpiLoc (http://epiloc.cs.queensu.ca/) and Plant-mPLoc (Chou and Shen, 2010) predicted that the subcellular localization of all tubulin proteins are Cytoplasmic/Chloroplast. TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and TOPCONS (Tsirigos et al. 2015) predicted that all tubulin proteins are located outside the membrane. This results indicated that the tubulin genes may synthesize the cytoskeleton in plant cells cytoplasm.

Chromosomal location, gene duplication, and collinearity relationships

Chromosomal location showed all tubulin genes scattered unequally on 24 chromosomes except At10 and Dt10 in G. hirsutum (Fig.3), which are mainly situated on the proximate ends of the chromosomes. At03 and Dt05 of G. hirsutum contained the most number of tubulin genes (7). By contrast, At02, At04, At06, Dt04, Dt07, Dt12 of G. hirsutum only included one or two of tubulin genes. There are deduced that genetic variation existed in the progress of evolution, and certified by this uneven distribution of the tubulin genes on the chromosomes. We further analyze the collinearity relationships for all tubulin genes on the chromosomes At and Dt of G. hirsutum with other cotton species. Among 42 tubulin genes on the chromosomes At of G. hirsutum, 33 tubulin had intergenomic homologous genes in chromosomes Dt of G. hirsutum (Fig.4); 40 tubulin homologous genes in G. arboretum (Fig. S3); among 48 tubulin genes on the chromosomes Dt of G. hirsutum, 48 tubulin had intergenomic homologous genes in G. raimondii (Fig. S4); and among all the 91 tubulin genes of G. hirsutum, 77 tubulin had intergenomic homologous genes in G. barbadense (Fig. S5), 91 tubulin homologous genes in G. darwinii and G. tomentosum(Fig. S6/7), respectively. Above results demonstrated that it may be an independent evolutionary process for At subgroup and Dt subgroup after the formation of allotetraploid, while it may be a same evolutionary direction for different allotetraploid cotton species upland cotton and wild cotton after the formation of allotetraploid.

Expression patterns of tubulin genes in different upland cotton

Although most tubulin superfamily genes have redundant functionality, they have similar functions together to promote cell elongation, supporting the close evolutionary relationships. To research the particular features of tubulin genes in ﬁber elongation and development, the expression patterns analyses was performed. Firstly, our RNA-sequencing data were used to detect the expression patterns of 98 tubulin genes in root, stem, leaf, ovule, and fiber tissues of G. hirsutum (J02-508 and ZRI-015) using a heatmap. The results showed that most α, β-tubulin genes had higher expression levels relative to γ-tubulin genes in all tissues, and the expression levels of most α, β-tubulin genes tubulins subfamilies give obvious intimation to functional divergence (Fig.5). Secondly, the expression levels of tubulin genes in the fiber development level had higher than other tissues e.g. Gh.A08G206600, Gh.D08G202200, Gh.A11G351700, Gh.D11G355300, Gh.A08G207900, Gh.A03G027200, Gh.D03G169300, Gh.A11G082000, Gh.D11G082500, Gh.A13G236900, Gh.A11G258900, Gh.A05G199500, Gh.D05G216200, Gh.D02G226100, Gh.A03G209100, Gh.A04G142400, Gh.D04G181300, Gh.A02G095500 and Gh.D02G097200, which may contribute to fiber development. Among them, only Gh.A03G027200, Gh.D03G169300 and Gh.A11G258900 had clear varies at distinct stages of ﬁber development in the two varieties, and they were significant differences at 15 DPA, which was a critical time for ﬁber length and strength in J02-508 compared to ZRI-015. Above results indicated the three genes may inhibit ﬁber length and strength in key stages of fiber development.

The expression levels of Gh.A03G027200, Gh.D03G169300 and Gh.A11G258900 in diﬀerent ﬁber developments for J02508 and ZRI015 were detected using qRT-PCR. As shown in Fig 6, the relative expression data revealed that the expression levels of Gh.A03G027200 and Gh.D03G169300 were the higher from 10 to 25 DPA in J02508 and ZRI015, and the expression levels of Gh.A11G258900 were higher from 3 to 15 DPA in the as well as twice in ZRI015 compared to J02508.

Cotton fibers are single-celled trichomes, which is important textile raw material. The tubulin proteins belong to microtubule proteins, which play an important role in the synthesis of the cytoskeleton (He et al. 2008). Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers (Schwarzerová et al. 2019). Microtubules are core element of the cytoskeleton and act a pivotal function in cellular migration, mitosis, mechanical stress, cell polarity, intracellular transport, cell division, and cell morphogenesis (He et al. 2008). This study determined 40 tubulin genes in A genomes, 43 tubulin genes in D genomes, 84 tubulin genes in AD₂ genomes, and 98 tubulin genes in AD₁ genomes. The results show that the loss of tubulin genes did not happen in allotetraploid G. hirsutum and G. barbadense, and some tubulin genes have been extended in G. hirsutum. The number of genes in allotetraploid cotton is twice as high as that of diploid cotton, which is difference with high rates of genes loss in allotetraploid plants (Zhang et al. 2015). Based on the previous reports about tubulin families in different plants (Pydiura et al. 2019), tubulin genes had been isolated into 3 subfamilies, our results were consistent with α, β and γ-tubulins. The previous reports showed that the structure and function of α, β-tubulins were quite similar, but that the γ-tubulins had great difference (Pydiura et al. 2019; Rao et al. 2016). In flax, expression analysis of tubulin genes showed that specific β-tubulin member could uphold vary microtubule functions such cell elongation, pollen tube development or cell wall thickening (Gavazzi et al. 2017; Rao et al. 2016). The amino acid sequences of eukaryotic tubulins are fairly well conserved but still display considerable differences in function (Hotta et al. 2016). The difference in the number of conserved motifs and exons between genes showed that functional diversity of tubulin may closely related to long evolution. For the tubulin gene members in cotton, we take particular mind to those that may play a key roles in fiber development.

To date, few tubulin genes were functionally characterized in plants, especially in the fiber-rich cotton. Combining the transcriptome expression of tubulin genes in J02508 and ZRI015, we find that some α, β-tubulins were specific expression in fiber development stages, while the γ-tubulin genes were low expression in all tissues. Research showed that some TUB genes were high expression in 10 DPA fiber after cotton flower (Li et al. 2002; He et al. 2008). Interestingly, Gh.A03G027200 and Gh.D03G169300 (belong to β-tubulins), Gh.A11G258900 (belong to α-tubulins) had clear varies in fiber development between J02508 and ZRI015, and they were significant differences at 15 DPA, which was a critical stage for fiber length and strength in J02508 compared to ZRI015. And the three genes may inhibit fiber length and strength in fiber development. Both Gh.A03G027200 and Gh.D03G169300 are homologous genes located in At and Dt of G. hirsutum respectively, which showed that they are together to perform the same function. However, the expression at homologous gene of Gh.A11G258900 is very low in all tissues of J02508 and ZRI015, which may indicate only Gh.A11G258900 perform function. All the above results indicate that the three tubulin genes may perform key functions for length and strength in fiber development.

Tubulin genes family acts a key role in cotton fiber development. Therefore, this study were first performed for a comprehensive analysis of tubulin genes family including phylogenetic tree, chromosomal location, collinearity, gene structure and expression patterns in cotton. The tubulin family genes are divided into three clades in the phylogenetic tree. The α, β-tubulin genes are highly conserved, yet γ-tubulin genes are less conservative among cotton and other plant species. The high expression of some α, β-tubulin gene in fiber development stage evidences that the demethylase family plays a significant role in cotton fiber development stage. The Gh.A03G027200, Gh.D03G169300 and Gh.A11G258900 had clear varies at a critical time for fiber length and strength in J02-508 compared to ZRI-015, indicating the three genes may inhibit fiber length and strength in key stages of fiber development. The results of our study built the foundation for excavating important functional genes and further studying the fiber length and strength mechanism of cotton.

MT: Microtubules; BLASTP: Basic Local Alignment Search Tool for Protein; FPKM: Fragments per Kilobase Million; DPA: Day(s) post-anthesis

Acknowledgments

Not applicable

Authors’ contributions

Chen BJ, Du XM and He SP conceived and designed the experiments; Zhao JJ, Fu GY, Pei XX, Pan ZE and Li HG performed the experiments and collected the data; Chen BJ obtained funding and Chen BJ, Ahmed H and Zhao JJ contributed reagents/materials/analysis tools; Chen BJ, Zhao JJ, Du XM and He SP revised the paper. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant No. 31621005).

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

Institute of Cotton Research, Chinese Academy of Agricultural Science (ICR, CAAS) / Research Base in Anyang Institute of Technology, State Key Laboratory of Cotton Biology, Anyang 455000, China.

Su JJ, Ma Q, Hao FS, Wang CX. Multi-locus genome-wide association studies of fiber-quality related traits in Chinese early-maturity upland cotton. Front Plant Sci. 2018, 9: 1169.
Gao ZY, Sun WJ, Wang J, Zhao CY, Zuo KJ. GhbHLH18 negatively regulates fiber strength and length by enhancing lignin biosynthesis in cotton fibers. Plant Sci. 2019, 286:7-16.
Gao P, Zhao PM, Wang J, Wang HY, Wu XM, Xia GX. Identification of genes preferentially expressed in cotton fibers: a possible role of calcium signaling in cotton fiber elongation. Plant Sci, 2007, 173(1), 61-69.
Wang M, Sun RR, Li C, Wang QL, Zhang BH. MicroRNA expression profiles during cotton (Gossypium hirsutum L) fiber early development. Sci Rep. 2017, 7: 44454.
Pydiura N, Pirko Y, Galinousky D, Postovoitova A, Yemets A, Kilchevsky A, Blume Y. Genome-wide identiﬁcation, phylogenetic classiﬁcation, and exon-intron structure characterization of the tubulin and actin genes in ﬂax (Linum usitatissimum). Cell Biol Int. 2019, 43(9):1010-1019.
Nieuwenhuis J and Brummelkamp TR. The tubulin detyrosination cycle: function and enzymes. Trends Cell Biol. 2019, 29(1): 80-92.
Findeisen P, Mühlhausen S, Dempewolf S, Hertzog J, Zietlow A, Carlomagno T, Kollmar M. Six subgroups and extensive recent duplications characterize the evolution of the eukaryotic tubulin protein family. Genome Biol Evol. 2014, 6(9): 2274-2288.
Zhao ZT, Liu HQ, Luo YP, Zhou SY, An L, Wang CF, Jin QJ, Zhou MG, Xu JR. Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life. Sci Rep. 2014, 4: 6746.
Jayaswal PK, Shanker A, Singh NK. Phylogeny of actin and tubulin gene homologs in diverse eukaryotic species. Indian J. Genet. 2019, 79(1): 284-291.
Mubeen H, Shoaib MW, Raza S. In silico analysis and comparison of alpha tubulin gene with other tubulin families. JABB. 2016, 7(4): 1-7.
Gavazzi F, Pigna G, Braglia L, Gianì S, Breviario D, Morello L. Evolutionary characterization and transcript profiling of β-tubulin genes in flax (Linum usitatissimum L.) during plant development. BMC Plant Biol. 2017, 17: 237.
Rao G, Zeng Y, He C, Zhang J. Characterization and putative post-translational regulation of α-and β-tubulin gene families in Salix arbutifolia. Sci Rep-UK. 2016, 6: 19258.
Hotta T, Fujita S, Uchimura S, Noguchi M, Demura T, Muto E, Hashimoto T. Affinity purification and characterization of functional tubulin from cell suspension cultures of Arabidopsis and tobacco. Plant Physiol. 2016, 170(3):1189-1205.
Miao H, Guo R, Chen J, Wang Q, Lee YRJ, Liu B. The γ-tubulin complex protein GCP6 is crucial for spindle morphogenesis but not essential for microtubule reorganization in Arabidopsis. P Natl A Sci India A. 2019, 116(52): 27115-27123.
Swamy PS, Hu H, Pattathil, S, Maloney VJ, Xiao H, Xue LJ, Chung JD, Johnson VE, Zhu YY, Peter GF, Hahn MG, Mansfield SD, Harding SA, Tsai CJ. Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus. J Exp Bot. 2015, 66(20): 6507-6518.
Sheoran IS, Koonjul P, Attieh J, Saini HS. Water-stress-induced inhibition of α-tubulin gene expression during growth, and its implications for reproductive success in rice. Plant Physiol Bioch. 2014, 80: 291-299.
Jöst M, Esfeld K, Burian A, Cannarozzi G, Chanyalew S, Kuhlemeier C, Assefa K, Tadele Z. Semi-dwarfism and lodging tolerance in tef (Eragrostis tef) is linked to a mutation in the α-Tubulin 1 gene. J Exp Bot. 2015, 66(3): 933-944.
Segami S, Kono I, Ando T, Yano M, Kitano H, Miura K, Iwasaki Y. Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice. Rice. 2012, 5(1): 4.
Gzyl J, Chmielowska-Bąk J, Przymusiński R, Gwóźdź EA. Cadmium affects microtubule organization and post-translational modifications of tubulin in seedlings of soybean (Glycine max L.). Front Plant Sci. 2015, 6: 937.
He XC, Qin YM, Xu Y, Hu CY, Zhu YX. Molecular cloning, expression profiling, and yeast complementation of 19 β-tubulin cDNAs from developing cotton ovules. J Exp Bot. 2008, 59(10): 2687-2695.
Swarbreck D, Wilks C, Lamesch P, Berardini TZ, Garcia-Hernandez M, Foerster H, Li D, Meyer T, Muller R, Ploetz L, Radenbaugh A, Singh S, Swing V, Tissier C, Zhang P, Huala E. The Arabidopsis information resource (TAIR): gene structure and function annotation. Nucl Acids Res. 2008, 36: D1009-14.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25(24): 4876-4882.
Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis. Mol Biol Evol.
2016, 33(7): 1870-4.
Zou C, Lu C, Shang H, Jing X, Cheng H, Zhang Y, Song G. Genome-wide analysis of the Sus gene family in cotton. J Integr Plant Biol. 2013, 55: 643-653.
Yang ZE, Ge XY, Yang ZR, Qin WQ, Sun GF, Wang Z, Li Z, Liu J, Wu J, Wang Y, Lu LL, Wang P, Mo HJ, Zhang XY, Li FG. Extensive intraspecific gene order and gene structural variations in upland cotton cultivars. Nat Commun. 2019, 10: 2989.
Schwarzerová K, Bellinvia E, Martinek J, SikorováL, Dostál V, Libusová L, Bokvaj P, Fischer L, Schmit AC, Nick P. Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway. Sci Rep. 2019, 9: 5725.
Zhang TZ, Hu Y, Jiang WK, Fang L, Guan XY, Chen JD, Zhang JB, Saski CA, Scheffler BE, Stelly DM, Hulse-Kemp AM, Wan Q, Liu BL, Liu CX, Wang S, Pan MQ, Wang YK, Wang DW, Ye WX, Chang LJ, Zhang WP, Song QX, Kirkbride RC, Chen XY, Dennis E, Llewellyn DJ, Peterson DJ, Thaxton P, Jones DC, Wang Q, Xu XY, Zhang H, Wu HT, Zhou L, Mei GF, Chen SQ, Tian Y, Xiang D, Li XH, Ding J, Zuo QY, Tao LN, Liu YC, Li J, Lin Y, Hui YY, Cao ZS, Cai CP, Zhu XF, Jiang Z, Zhou BL, Guo WZ, Li RQ, Chen ZJ. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for ﬁber improvement. Nat Biotechnol. 2015, 33(5):531-7.
Chen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH, Xia R. TBtools - an integrative toolkit developed for interactive analyses of big biological data. bioRxiv. 2020, doi: https://doi.org/10.1101/289660.
Ray DL and Johnson JC. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR. BMC Research Notes. 2014, 7: 304.
Šutković J, Gawwad MRA. In silico prediction of three-dimensional structure and interactome
analysis of Tubulin α subfamily of Arabidopsis thaliana. Network Biology. 2014, 4(2): 47-57.
Blume YB, Krasylenko YA, Demchuk OM, Yemets AI. Tubulin tyrosine nitration regulates microtubule organization in plant cells. Front Plant Sci. 2013, 4: 530.
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009, 19(9):1639-645.
Pierleoni A, Martelli PL, Fariselli P, Casadio R. BaCelLo: a balanced subcellular localization predictor. Bioinformatics. 2006, 22(14): e408-e416.
Chou KC, Shen HB. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE. 2010, 5: e11335.
Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res. 2015, 43(W1): W401-W407.
Li XB, Fan XP, Wang XL, Cai L, Yang WC. The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell. 2005, 17: 3.
Li XB, Cai L, Cheng NH, Liu JW. Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. Plant Physiol. 2002, 130(2): 666-674.

Due to technical limitations, table 1 and table 2 are only available as a download in the Supplemental Files section.

Table1andTable2.xlsx
Table 1: Primers used in qRT-PCR detection of three key Tubulin genes from G. hirsutum
Table 2: Basic characteristic of tubulin family genes in cotton genome
Fig.S1.jpg
Conserved domain analysis of tubulin gene family.
Fig.S2.jpg
Conserved site analysis of tubulin gene family.
Fig.S3.jpg
Collinearity of tubulin genes in Goppypium arboretum and Goppysium hisutum species.
Fig.S4.jpg
Collinearity of tubulin genes in Goppypium raimondii and Goppysium hisutum species.
Fig.S5.jpg
Collinearity of tubulin genes in Goppysium hisutum and Goppysium bartadense species.
Fig.S6.jpg
Collinearity of tubulin genes in Goppysium hisutum and Goppysium tomentosum species.
Fig.S7.jpg
Collinearity of tubulin genes in Goppysium hisutum and Goppysium darwinii species.

Download PDF

Journal Publication

published 13 Jul, 2021

Read the published version in Journal of Cotton Research →

Editorial decision: Major revision
14 Mar, 2021
Reviewer #3 agreed at journal
05 Mar, 2021
Review #3 received at journal
05 Mar, 2021
Review #2 received at journal
16 Jan, 2021
Reviewer #2 agreed at journal
29 Dec, 2020
Reviewers invited by journal
13 Dec, 2020
Reviewer #1 agreed at journal
13 Dec, 2020
Review #1 received at journal
13 Dec, 2020
Editor assigned by journal
02 Dec, 2020
Submission checks completed at journal
02 Dec, 2020
Editor invited by journal
02 Dec, 2020
First submitted to journal
30 Nov, 2020

You are reading this latest preprint version

Identification and Expression Analysis of Tubulin Gene Family in Upland Cotton

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Plant Material and Nucleic Acid Extraction

Sequence retrieval and phylogenetic analysis

Gene structure, chromosomal mapping and collinearity analysis

Gene Expression Analysis

Result

Phylogenetic analysis of the tubulin protein family

Gene structure conserved motifs analysis

Chromosomal location, gene duplication, and collinearity relationships

Expression patterns of tubulin genes in different upland cotton

Discussion

Conclusion

Abbreviations

Declarations

Acknowledgments

Authors’ contributions

Funding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

Author details

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1