棉花微管蛋白<i>Togaram1</i>基因的克隆及表达分析

doi:10.7668/hbnxb.20195290

摘要/Abstract

摘要：

棉纤维细胞中的微管及微管结合蛋白对细胞内物质运输及形态建成具有重要调控作用。TOG结构域是调节微管动力学的几个蛋白质家族的重要功能组分,CLASPs则是植物中特有的一类微管结合蛋白,其表达对于微管的动态调整和功能行使十分重要。为了研究Togaram1基因与棉花纤维发育及纤维品质形成的关系,以陆地棉系9和海岛棉新海16为研究材料,克隆获得GhTogaram1与GbTogaram1基因,并对其进行生物信息学分析,同时,利用qRT-PCR技术分析Togaram1基因在不同材料及不同纤维发育时期的表达模式。结果显示,GhTogaram1与GbTogaram1的CDS序列全长均为918 bp,均编码305个氨基酸,但陆地棉和海岛棉之间存在核苷酸和氨基酸序列差异。生物信息学分析表明,Togaram1蛋白属于亲水性蛋白,具有TOG结构域和CLASP-N端结构域,定位于细胞核中。系统进化树和多序列比对表明,GhTogaram1和海岛棉属于同一分支,相似性为99.34%,与二者关系最近的是黄褐棉。qRT-PCR结果表明,Togaram1基因在2种材料纤维发育不同时期(0,5,25 d)相对表达量具有极显著差异;另外,Togaram1基因在HL群体极端子代中纤维发育5 d和10 d具有极显著差异,其中短纤维株系HL-78表达量最高。综上,Togaram1基因在不同棉花材料开花后5 d纤维中优势表达,表明Togaram1基因可能在棉花纤维伸长期发挥作用。随着开花天数的增加,Togaram1基因表达量下降,推测可能是受油菜素甾醇的影响。对Togaram1基因在棉纤维中进行初步的功能验证,为后续Togaram1基因在棉花纤维发育中的研究奠定了基础。

关键词: 棉花, 棉纤维, 基因克隆, Togaram1, 表达分析

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

中图分类号:

李生梅, 李静, 庞博, 张茹, 耿世伟, 陈佳林, 宋武, 高文伟. 棉花微管蛋白Togaram1基因的克隆及表达分析[J]. 华北农学报, 2025, 40(4): 76-84. doi: 10.7668/hbnxb.20195290.

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.

http://www.hbnxb.net/CN/Y2025/V40/I4/76

图/表 15

表1 生物信息学预测网站

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/

图1 连接T载菌液PCR产物 M.DL2000 Marker;1.GhTogaram1基因;2.GbTogaram1基因。

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

图2 GhTogaram1与GbTogaram1基因核苷酸序列对比序列上.GhTogaram1;序列下.GbTogaram1。

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

图3 GhTogaram1与GbTogaram1编码蛋白氨基酸序列对比

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

表2 GhTogaram1和GbTogaram1蛋白理化性质

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

图4 GhTogaram1与GbTogaram1编码蛋白的亲/疏水性预测

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

图5 GhTogaram1与GbTogaram1编码蛋白的信号肽预测

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

图6 GhTogaram1与GbTogaram1编码蛋白的亚细胞定位预测

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

图7 GhTogaram1与GbTogaram1编码蛋白的结构域预测 A.GhTogaram1蛋白;B.GbTogaram1蛋白。图8-9同。

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

图8 GhTogaram1与GbTogaram1编码蛋白的二级结构

Fig.8 Secondary structure of GhTogaram1 and GbTogaram1 encoded proteins

图9 GhTogaram1与GbTogaram1蛋白的三级结构

Fig.9 Tertiary structure of GhTogaram1 and GbTogaram1 proteins

图10 不同植物Togaram1氨基酸序列的多重比对

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

图11 不同植物Togaram1氨基酸序列的系统进化树

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

图12 Togaram1基因在纤维组织的表达量不同大写字母表示差异在P<0.01具有极显著性。图13同。

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

图13 Togaram1基因在极端材料纤维组织的表达量

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

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棉花微管蛋白Togaram1基因的克隆及表达分析

Cloning and Expression Analysis of Tubulin Togaram1 Gene in Cotton

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