陆地棉GHWAT1-35基因的克隆及亚细胞定位

doi:10.6048/j.issn.1001-4330.2024.06.002

摘要/Abstract

摘要：

【目的】研究陆地棉GHWAT1-35基因在棉花纤维发育过程中的作用。【方法】选用陆地棉系9花后不同时期的纤维为材料,克隆GHWAT1-35基因的全长cDNA序列,并进行生物信息学、实时荧光定量(qRT-PCR)分析及亚细胞定位。【结果】该基因全长1 125 bp,编码374个氨基酸,相对分子量为40.231 kDa,理论等电点为8.74。GHWAT1-35蛋白与亚洲棉亲缘关系最近。GHWAT1-35基因在开花后15 d表达量显著升高,亚细胞定位预测表明该蛋白定位于细胞质膜上。将GHWAT1-35基因与pCAMIA1300-35S-YFP载体重组,构建融合表达载体,利用冻融法转化农杆菌,注射到烟草后观察到GHWAT1-35蛋白定位在细胞质膜上。【结论】陆地棉系9 GHWAT1-35基因在开花后15和20 DAP的表达量显著高于其他时期,pC1300::35S-WAT-YFP融合蛋白定位于细胞质膜上。

关键词: 陆地棉; WAT1; 基因克隆; 生物信息学; 亚细胞定位

Abstract:

【Objective】 To explore the role of GHWAT1-35 gene in the development of cotton fibers. 【Methods】 In this study, the fiber at different developmental stages post-anthesis of Gossypium hirsutum variety Xi9 were used as materials.The full-length cDNA sequence of the GHWAT1-35 gene was successfully cloned and subjected to bioinformatics analysis, real-time fluorescence quantitative (qRT-PCR) analysis, and subcellular localization. 【Results】 The length of the GHWAT1-35 gene was 1125 bp, encoding 374 amino acids, with a relative molecular weight is 40.23 kDa and a theoretical isoelectric point of 8.74.Protein multiple sequence alignment and construction of a systematic evolutionary tree analysis showed that the GHWAT1-35 protein was most closely related to the Gossypium arboreum.The expression of the GHWAT1-35 gene significantly increased at 15 days after flowering, and subcellular localization predicted that the protein was located on the cytoplasmic membrane.The GHWAT1-35 gene was recombined with the pCAMIA1300-35S-YFP vector to construct a fusion expression vector, which was then transformed into Agrobacterium using the freeze-thaw method and after being injected into tobacco.The GHWAT1-35 protein was observed to be located on the cytoplasmic membrane. 【Conclusion】 The GHWAT1-35 gene plays an important role in fiber development, providing a foundation for further exploration of its biological function in Gossypium hirsutum.

Key words: Gossypium hirsutum; WAT1; gene cloning; bioinformatics; subcellular localization

中图分类号:

S562

马尚洁, 李生梅, 杨涛, 王红刚, 赵康, 庞博, 高文伟. 陆地棉GHWAT1-35基因的克隆及亚细胞定位[J]. 新疆农业科学, 2024, 61(6): 1310-1317.

MA Shangjie, LI Shengmei, YANG Tao, WANG Honggang, ZHAO Kang, PANG Bo, GAO Wenwei. Cloning and subcellular localization of the GHWAT1-35 gene in Gossypium hirsutum[J]. Xinjiang Agricultural Sciences, 2024, 61(6): 1310-1317.

图/表 13

图1 GHWAT1-35基因电泳图注: M: DL2000 DNA Marker; 1:GHWAT1-35基因扩增产物

Fig.1 GHWAT1-35 gene electrophoresis Note: M: DL2000 DNA Marker; 1: PCR product of GHWAT1-35 gene

图2 GHWAT1-35蛋白亲水性预测

Fig.2 Prediction of the hydrophilicity of GHWAT1-35 protein

图3 GHWAT1-35蛋白磷酸化位点预测

Fig.3 Prediction of phosphorylation site of GHWAT1-35 protein

图4 GHWAT1-35蛋白跨膜区预测

Fig.4 Prediction of transmembrane domains of GHWAT1-35 protein

图5 GHWAT1-35蛋白信号肽预测

Fig.5 GHWAT1-35 protein signal peptide prediction

图6 不同物种WAT1蛋白氨基酸多序列比度

Fig.6 Multiple alignments of amino acid sequence of WAT1 form different specie

图7 不同物种 WAT1 蛋白的系统进化

Fig.7 Phylogenetic analysis of WAT1 proteins from different species

图8 GHWAT1-35蛋白二级结构的预测

Fig.8 Prediction of secondary structure of GHWAT1-35 protein

图9 GHWAT1-35蛋白三级结构的预测

Fig.9 Prediction of tertiary structure of GHWAT1-35 protein

图10 GHWAT1-35蛋白亚细胞定位预测

Fig.10 Subcellular localization prediction of GHWAT1-35 protein

表1 GHWAT1-35 启动子顺式元件

Tab.1 Analysis of cis-elements GHWAT1-35 promoter

顺式作用元件 Cis- elements	序列 Sequence (5'-3')	功能 Function	数量 No.
CAAT-box	CAAT	控制着转录起始的频率	19
TATA-box	TATA	转录开始的-30左右的核心启动子元件	5
STRE	AGGGG	应激反应元件	2
MYB	CAACAG	MYB响应元件;激素信号转导和非生物胁迫响应	4
CGTCA-motif	CGTCA	参与MeJA反应的顺式作用调节元件	2
GT1-motif	GGTTAA	光响应元件	1
P-box	CCTTTTG	赤霉素响应元件	1
ARE	AAACCA	厌氧诱导所必需的顺式作用调节元件	1
O2-site	GATGATGTGG	参与玉米醇溶蛋白代谢途径顺式作用元件	1

图11 GHWAT1-35基因的表达

Fig.11 Gene expression analysis of GHWAT1-35

图12 GhWAT1-35亚细胞定位

Fig.12 GhWAT1-35 subcellular localization

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