Cloning and Expression Analysis of Tubulin Togaram1 Gene in Cotton

doi:10.7668/hbnxb.20195290

Abstract

Abstract:

Microtubules and microtubule-binding proteins in cotton fibroblasts play an important role in regulating intracellular material transport and morphogenesis.The TOG domain is an important functional component of several protein families that regulate microtubule dynamics,and CLASPs are a unique class of microtubule-binding proteins in plants,whose expression is very important for the dynamic adjustment and functional exercise of microtubules.In order to study the relationship between Togaram1 gene and cotton fiber development and fiber quality formation,GhTogaram1 and GbTogaram1 genes were cloned from upland cotton Line 9 and sea island cotton Xinhai 16,and their bioinformatics analysis was conducted.Meanwhile,qRT-PCR was used to analyze the expression patterns of Togaram1 gene in different materials and different fiber development stages.The results showed that the total length of CDS sequence of GhTogaram1 and GbTogaram1 was 918 bp,which encoded 305 amino acids,but there were nucleotide and amino acid sequence differences between upland cotton and sea island cotton.Bioinformatics analysis showed that Togaram1 protein was a hydrophilic protein with TOG domain and CLASP-N-terminal domain,and was localized in the nucleus.The phylogenetic tree and multiple sequence comparison showed that GhTogaram1 and Gossypium barbadense belonged to the same cladisticbranch,the similarity was 99.34%,and the closest relation was Gossypium mustelinum.The results of qRT-PCR showed that the relative expression levels of Togaram1 gene were significantly different at different stages of fiber development(0,5,25 d)between the two materials.In addition,there were significant differences in the fiber development at 5 and 10 d of Togaram1 gene in the extreme offspring of the HL population,and the expression level of HL-78 was the highest in the short fiber line.In conclusion,Togaram1 gene was predominantly expressed in the fibers of different cotton materials 5 d after flowering,indicating that Togaram1 gene may play a role in cotton fiber elongation.With the increase of flowering days,the expression of Togaram1 gene decreased,which may be due to the influence of brassinsterol.The preliminary functional verification of Togaram1 gene in cotton fiber laid a foundation for the subsequent study of Togaram1 gene in cotton fiber development.

Key words: Cotton, Cotton fiber, Gene cloning, Togaram1, Expression analysis

CLC Number:

LI Shengmei, LI Jing, PANG Bo, ZHANG Ru, GENG Shiwei, CHEN Jialin, SONG Wu, GAO Wenwei. Cloning and Expression Analysis of Tubulin Togaram1 Gene in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 76-84. doi: 10.7668/hbnxb.20195290.

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Figures/Tables 15

Tab.1 Bioinformatics prediction websites

数据库 Database	预测功能 Forecasting capabilities	网址 Website
ProtParam	理化性质	http://web.expasy.org/protparam/
ProtScale	亲疏水性	http://prosite.expasy.org/
LOC TREE3(5R)	亚细胞定位	https://rostlab.org/services/loctree3/
SignalP-5	信号肽	http://www.cbs.dtu.dk/services/SignalP
Interproscan	基因结构域	https://www.plob.org/tag/interproscan
SOPMA	二级结构	https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma
I-TASSER	三级结构	https://zhanggroup.org/I-TASSER/

Fig.1 PCR product of bacterial solution after connecting the T carrier M.DL2000 Marker;1.GhTogaram1 gene;2.GbTogaram1 gene.

Fig.2 Comparison of nucleotide sequences of GhTogaram1 and GbTogaram1 genes Sequence above.GhTogaram1;Sequence below.GbTogaram1.

Fig.3 Comparison of amino acid sequences of proteins encoded by GhTogaram1 and GbTogaram1

Tab.2 Physicochemical properties of GhTogaram1 and GbTogaram1 proteins

类型Type	GhTogaram1	GbTogaram1
预测分子式Predictive molecular formula	C₁₄₈₅H₂₄₀₇N₄₀₁O₄₅₄S₁₁	C₁₄₈₇H₂₄₁₀N₄₀₀O₄₅₂S₁₁
原子总数/个Total number of atoms	4 758	4 760
分子质量/ku Molecular weight	33.50	33.47
理论等电点Theoretical pI	7.62	7.62
带负电荷的残基总数(Asp+Glu)/个	37	37
Total number of negatively charged residues(Asp+Glu)
带正电荷的残基总数(Arg+Lys)/个	38	38
Total number of positively charged residues(Arg+Lys)
不稳定系数Instability index	42.23	41.95
脂肪系数Aliphatic index	90.20	91.48
亲水性平均系数(GRAVY) Grand average of hydropathicity(GRAVY)	-0.281	-0.257

Fig.4 Prediction of hydrophilicity/hydrophobicity of GhTogaram1 and GbTogaram1 encoded proteins

Fig.5 Signal peptide prediction of GhTogaram1 and GbTogaram1 encoded proteins

Fig.6 Prediction of subcellular localization of GhTogaram1 and GbTogaram1 encoded proteins

Fig.7 Domain prediction of GhTogaram1 and GbTogaram1 encoded proteins A.GhTogaram1 protein;B.GbTogaram1 protein.The same as Fig.8-9.

Fig.8 Secondary structure of GhTogaram1 and GbTogaram1 encoded proteins

Fig.9 Tertiary structure of GhTogaram1 and GbTogaram1 proteins

Fig.10 Multiple alignment of Togaram1 amino acid sequences from different plants

Fig.11 Phylogenetic tree of Togaram1 amino acid sequences from different plants

Fig.12 Expression of Togaram1 gene in fibrous tissue Different letters show significant difference at P<0.01 level.The same as Fig.13.

Fig.13 Expression of Togaram1 gene in fibrous tissue of extreme materials

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