Cloning and subcellular localization of the GHWAT1-35 gene in Gossypium hirsutum

doi:10.6048/j.issn.1001-4330.2024.06.002

Abstract

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

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

CLC Number:

S562

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.

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.

Figures/Tables 13

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

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

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

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

Fig.5 GHWAT1-35 protein signal peptide prediction

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

Fig.7 Phylogenetic analysis of WAT1 proteins from different species

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

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

Fig.10 Subcellular localization prediction of GHWAT1-35 protein

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

Fig.11 Gene expression analysis of GHWAT1-35

Fig.12 GhWAT1-35 subcellular localization

References 30

[1]	喻树迅, 范术丽, 王寒涛, 等. 中国棉花高产育种研究进展[J]. 中国农业科学, 2016, 49(18): 3465-3476. DOI
	YU Shuxun, FAN Shuli, WANG Hantao, et al. Progresses in research on cotton high yield breeding in China[J]. Scientia Agricultura Sinica, 2016, 49(18): 3465-3476. DOI
[2]	丁玉, 何安华, 汪丽华. 我国棉花产业供求现状与趋势[J]. 重庆社会科学, 2012,(7): 80-85.
	DING Yu, HE Anhua, WANG Lihua. The current situation and trend of the supply and demand of the cotton industry[J]. Chongqing Social Sciences, 2012,(7): 80-85.
[3]	郭三堆, 王远, 孙国清, 等. 中国转基因棉花研发应用二十年[J]. 中国农业科学, 2015, 48(17): 3372-3387. DOI
	GUO Sandui, WANG Yuan, SUN Guoqing, et al. Twenty years of research and application of transgenic cotton in China[J]. Scientia Agricultura Sinica, 2015, 48(17): 3372-3387. DOI
[4]	Busov V B, Johannes E, Whetten R W, et al. An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein[J]. Planta, 2004, 218(6): 916-927.
[5]	Ranocha P, Denancé N, Vanholme R, et al. Walls are thin?1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers[J]. The Plant Journal, 2010, 63(3): 469-483.
[6]	Mitsuda N, Iwase A, Yamamoto H, et al. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis[J]. The Plant Cell, 2007, 19(1): 270-280.
[7]	Ranocha P, Dima O, Nagy R, et al. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis[J]. Nature Communications, 2013, 4: 2625.
[8]	彭方林. 拟南芥类结瘤素基因At1g01070的表达模式及功能的初步研究[D]. 新乡: 河南师范大学, 2015.
	PENG Fanglin. Preliminary Study on Expression Pattern and Function of Nodoid Gene At1g01070 in Arabidopsis thaliana[D]. Xinxiang: Henan Normal University, 2015.
[9]	Ju F Y, Liu S D, Zhang S P, et al. Transcriptome analysis and identification of genes associated with fruiting branch internode elongation in upland cotton[J]. BMC Plant Biology, 2019, 19(1): 415. DOI PMID
[10]	Tang Y, Zhang Z N, Lei Y, et al. Cotton WATs modulate SA biosynthesis and local lignin deposition participating in plant resistance against Verticillium dahliae[J]. Frontiers in Plant Science, 2019, 10: 526. DOI PMID
[11]	王海霞, 崔大勇. 陆生植物生长素合成的主要途径及调控[J]. 生物学教学, 2015, 40(9): 2-5.
	WANG Haixia, CUI Dayong. Main ways and regulation of auxin synthesis in terrestrial plants[J]. Biology Teaching, 2015, 40(9): 2-5.
[12]	贺新强, 崔克明. 植物细胞次生壁形成的研究进展[J]. 植物学通报, 2002,(5):513-522.
	HE Xinqiang, CUI Keming. Progress in study of secondary wall formation in plants[J]. Chinese Bulletin of Botany, 2002,(5):513-522.
[13]	Hanika K, Schipper D, Chinnappa S, et al. Impairment of tomato WAT1 enhances resistance to vascular wilt fungi despite severe growth defects[J]. Frontiers in Plant Science, 2021, 12: 721674.
[14]	刘嘉鹏, 屈蒙蒙, 孙雪丽, 等. 香蕉WAT1基因的鉴定及表达分析[J]. 果树学报, 2020, 37(5): 645-658.
	LIU Jiapeng, QU Mengmeng, SUN Xueli, et al. Identification and expression analysis of WAT1 genes in banana[J]. Journal of Fruit Science, 2020, 37(5): 645-658.
[15]	Ladwig F, Stahl M, Ludewig U, et al. Siliques are Red1 from Arabidopsis acts as a bidirectional amino acid transporter that is crucial for the amino acid homeostasis of siliques[J]. Plant Physiology, 2012, 158(4): 1643-1655.
[16]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI PMID
[17]	孔佑宾, 王冰, 张华, 等. 植物蛋白融合HA标签通用载体构建与应用[J]. 中国农业科技导报, 2017, 19(6): 131-137.
	KONG Youbin, WANG Bing, ZHANG Hua, et al. Construction and amplification of protein fusion HA tag universal expression vector in plant[J]. Journal of Agricultural Science and Technology, 2017, 19(6): 131-137. DOI
[18]	张边江, 陈全战. 质粒导入不同种农杆菌冻融法的探讨[J]. 湖北农业科学, 2007, 46(3): 329-331.
	ZHANG Bianjiang, CHEN Quanzhan. Study on the freeze-thaw method of transforming plasmid DNA into two different Agrobacterium tumefaciens[J]. Hubei Agricultural Sciences, 2007, 46(3): 329-331.
[19]	马春泉, 孙培琳, 李海英. BvM14-GAI基因的克隆及亚细胞定位[J]. 中国农学通报, 2020, 36(16): 28-33. DOI
	MA Chunquan, SUN Peilin, LI Haiying. BvM14-GAI gene: cloning and subcellular localization[J]. Chinese Agricultural Science Bulletin, 2020, 36(16): 28-33. DOI
[20]	孙蔓莉, 孟玉玲, 张强, 等. 农杆菌介导的烟草瞬时表达影响因素研究[J]. 西北农业学报, 2015, 24(1): 161-165.
	SUN Manli, MENG Yuling, ZHANG Qiang, et al. Optimization of Agrobacterium-mediated transient assay of gene expression in Nicotiana tobacum[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2015, 24(1): 161-165.
[21]	Nelson B K, Cai X, Nebenführ A. A multicolored set of in?vivo organelle markers for co-localization studies in Arabidopsis and other plants[J]. The Plant Journal, 2007, 51(6): 1126-1136.
[22]	李濯雪, 陈信波. 植物诱导型启动子及相关顺式作用元件研究进展[J]. 生物技术通报, 2015, 31(10): 8-15. DOI
	LI Zhuoxue, CHEN Xinbo. Research advances on plant inducible promoters and related Cis-acting Elements[J]. Biotechnology Bulletin, 2015, 31(10): 8-15. DOI
[23]	Farquharson K L. Probing the role of auxin in wood formation[J]. The Plant Cell, 2008, 20(4): 822.
[24]	Sousa C, Johansson C, Charon C, et al. Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex[J]. Molecular and Cellular Biology, 2001, 21(1): 354-366. DOI PMID
[25]	王晶晶. 棉花类结瘤素基因GhMtN21-31棉花黄萎病抗性中的功能研究[D]. 南京: 南京农业大学, 2022.
	WANG Jingjing. Functional analysis of a nodulin-like gene ghmtn21-31 in cotton resistance against verticillium wilt[D]. Nanjing: Nanjing Agricultural University, 2022.
[26]	邢浩然, 刘丽娟, 刘国振. 植物蛋白质的亚细胞定位研究进展[J]. 华北农学报, 2006, 21(S2): 1-6. DOI
	XING Haoran, LIU Lijuan, LIU Guozhen. Advancement of protein subcellular localization in plants[J]. Acta Agriculturae Boreali-Sinica, 2006, 21(S2): 1-6.
[27]	张松, 夏学峰, 沈金城, 等. 基于序列保守性和蛋白质相互作用的真核蛋白质亚细胞定位预测[J]. 生物化学与生物物理进展, 2008, 35(5): 531-535.
	ZHANG Song, XIA Xuefeng, SHEN Jincheng, et al. Eukaryotic protein subcellular localization prediction based on sequence conservation and protein-protein interaction[J]. Progress in Biochemistry and Biophysics, 2008, 35(5): 531-535.
[28]	谢宏, 李舒文, 董迪, 等. 蒺藜苜蓿MtDWF1基因克隆、亚细胞定位及表达特征分析[J]. 草地学报, 2023, 31(4): 984-991. DOI
	XIE Hong, LI Shuwen, DONG Di, et al. Cloning, subcellular localization and expression pattern of MtDWF1 gene in Medicago truncatula[J]. Acta Agrestia Sinica, 2023, 31(4): 984-991. DOI
[29]	Mansoor S, Paterson A H. Genomes for jeans: cotton genomics for engineering superior fiber[J]. Trends in Biotechnology, 2012, 30(10): 521-527. DOI PMID
[30]	上官小霞, 曹俊峰, 杨琴莉, 等. 棉花纤维发育的分子机理研究进展[J]. 棉花学报, 2022, 34(1): 33-47. DOI
	SHANGGUAN Xiaoxia, CAO Junfeng, YANG Qinli, et al. Research progress on the molecular mechanism of cotton fiber development[J]. Cotton Science, 2022, 34(1): 33-47.