Cloning and bioinformatics analysis of β-carotene isomerase GbD27-6 gene in sea island cotton

doi:10.6048/j.issn.1001-4330.2023.12.002

Xinjiang Agricultural Sciences ›› 2023, Vol. 60 ›› Issue (12): 2869-2877.DOI: 10.6048/j.issn.1001-4330.2023.12.002

• Crop Genetics and Breeding·Germplasm Resources·Molecular Genetics·Soil Fertilizer • Previous Articles Next Articles

Cloning and bioinformatics analysis of β-carotene isomerase GbD27-6 gene in sea island cotton

LIU Chenxi¹(), ZHU Yuting¹, ZHOU Qiang¹, CHEN Jin¹, ZHAO Wenjie¹, ZHENG Kai¹^,²()

1. Key Laboratory of Agricultural Biotechnology/College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, China
2. Postdoctoral Mobile Station of Xinjiang Agricultural University, Urumqi 830052, China

Received:2023-04-09 Online:2023-12-20 Published:2024-01-03
Correspondence author: ZHENG Kai(1988-), male, Xinyang, Henan Province, associate professor, doctoral and master tutor, research direction: cotton molecular breeding, (E-mail)zhengkai555@126.com
Supported by:
Major S & T Project of Xinjiang Uygur Autonomous Region(2022A03004-2);Urumqi "Young Doctoral Science and Technology Talent Training" Project(2019KJTXM);Financial Support Project of Xinjiang Uygur Autonomous Region of Department of Agriculture and Rural Affairs(2022MH-01);Student Innovation Project of Xinjiang Agricultural University(dxscx2021018)

海岛棉β-胡萝卜素异构酶GbD27-6基因克隆及表达分析

刘晨曦¹(), 朱雨婷¹, 周强¹, 陈瑾¹, 赵文杰¹, 郑凯¹^,²()

1.新疆农业大学农学院/农业生物技术重点实验室,乌鲁木齐 830052
2.新疆农业大学博士后流动站,乌鲁木齐 830052

通讯作者: 郑凯(1988-),男,河南信阳人,讲师,博士,硕士生导师,研究方向为棉花分子育种,(E-mail)zhengkai555@126.com
作者简介:刘晨曦(2001-),男,山东临沂人,研究方向为种子科学与工程,(E-mail)1176666997@qq.com
基金资助:
新疆维吾尔自治区重大科技专项(2022A03004-2);乌鲁木齐“青年博士科技人才培养”项目(2019KJTXM);新疆维吾尔自治区农业农村厅财政项目(2022MH-01);新疆农业大学大学生创新项目(dxscx2021018)

Abstract

Abstract:

【Objective】To study the effect of GbD27-6 gene on branch and lateral branch growth of sea island cotton. This study provides a basic theory for further understanding the role of GbD27 gene in intracellular transport and its participation in epidermal cell processes during fiber development.【Methods】In this study, the key gene GbD27-6 in the synthesis pathway of Strigolactones (SLs) was cloned from the fibrous material of Gossypium barbadense L. Pimas-7 15 d, and the expression level of GbD27-6 was analyzed by using the bioinformatics website and qRT-PCR.【Results】GbD27-6 gene 816 bp encoded 271 amino acid protein, molecular formula is was C₁₃₃₅H₂₁₃₄N₃₅₀O₃₈₉S₂₄, molecular weight is was about 30.081 18 kDa, isoelectric point is was 8.40, it which belonged to DUF4033 superfamily protein and was unstable hydrophobic protein. The results of subcellular localization showed that GbD27-6 gene was located on cell membrane. Real -time fluorescence quantitative analysis showed that GbD27-6 gene was expressed in petal bracts and anthers of Gossypium barbadense L. and the expression level was the highest in bracts and the lowest in petals. The expression level was the highest on the 20th day of fiber development and the lowest on the 10th day of fiber development.【Conclusion】The findings showed that GbD27-6 gene was differentially expressed in fibers.

Key words: Gossypium barbadense L; GbD27-6; gene cloning; bioinformatics

摘要：

【目的】研究GbD27-6基因在海岛棉分枝和侧枝生长中的影响作用,为分析GbD27基因在胞内运输作用及在纤维发育中参与表皮细胞突起研究提供参考。【方法】以海岛棉Pimas-7 在15 d的纤维材料为模板,克隆独脚金内酯(SLs)合成通路中的关键基因GbD27-6,利用生物信息学分析网站分析该基因,并利用qRT-PCR技术分析其基因表达量的差异性。【结果】GbD27-6基因全长816 bp,编码271个氨基酸,蛋白分子式为C₁₃₃₅H₂₁₃₄N₃₅₀O₃₈₉S₂₄,分子质量大约为30.081 18 kDa,等电点为8.40,属于DUF4033超家族蛋白成员,且为不稳定疏水性蛋白。GbD27-6基因定位在细胞膜,GbD27-6基因在海岛棉花瓣、苞叶、花药中均有表达,其中在苞叶中表达量最高,在花瓣中表达量最低;而在不同发育时期的纤维中均有不同程度的表达,其中在纤维发育20 d的表达量最高,在10 d时表达量最低。【结论】GbD27-6基因在纤维中存在差异表达。

关键词: 海岛棉, GbD27-6, 基因克隆, 生物信息学

CLC Number:

S562

LIU Chenxi, ZHU Yuting, ZHOU Qiang, CHEN Jin, ZHAO Wenjie, ZHENG Kai. Cloning and bioinformatics analysis of β-carotene isomerase GbD27-6 gene in sea island cotton[J]. Xinjiang Agricultural Sciences, 2023, 60(12): 2869-2877.

刘晨曦, 朱雨婷, 周强, 陈瑾, 赵文杰, 郑凯. 海岛棉β-胡萝卜素异构酶GbD27-6基因克隆及表达分析[J]. 新疆农业科学, 2023, 60(12): 2869-2877.

Figures/Tables 16

Tab.1 Primer sequence

基因名称 Gene name	引物名称 Primer name	引物序列 Primer sequence(5'-3')
GbD27-6	qGbD27-6F	ATGAAAATGATGCCTCTGGCTCTGA
GbD27-6	qGbD27-6R	TTACGCGTTTGGACATTTCATCACAT
UBQ7	UBQ7-F	GACCTACACCAAGCCCAAGAAG
UBQ7	UBQ7-R	TGAGCCCACACTTACCACAATAGT

Tab.2 Bioinformatics analysis software

软件名称 Software name	网址 URL	作用 Effect
Genbank	https://www.ncbi.nlm.nih.gov/genbank/	CDS序列分析
ExPasy	http://web.expasy.prg/protparam/	理化性质预测
WoLF PSORT	https://wolfpsort.hgc.jp/	亚细胞定位预测
SOPMA	http://npsa-pbil.ibcp.fr/cgi-bin/npsa	蛋白质二级、三级结构预测
SOPMA	_automat.pl page=npsa_sopma.html	蛋白质二级、三级结构预测
SignaIP	http://www.cbs.dtu.dk/services/SignaIP	蛋白质信号肽预测分析
TMHMM	http://www.cbs.dtu.dk/services/TMHMM/	蛋白质跨膜区分析
SMART	http://smart.embl-heidelberg.de/	保守结构域分析
NetPhos	http://www.cbs.dtu.dk/services/NetPhos	磷酸化位点分析
MEME	https://meme-suite.org/meme/	motif分析
MEGA	https://megasoftware.net/	多序列比对、进化树分析

Fig.1 Electrophoresis diagram of GbD27-6 gene of sea island cotton M:DL2000 DNA maker;1:Amplified product of GbD27-6 gene

Fig.2 Electrophoresis diagram of GbD27-6 gene bacterial fluid of sea island cotton Note:M:DL2000 DNA maker;2:Positive identification of the bacterial fluid of GbD27-6 gene

Fig.3 Hydrophilic and hydrophobic analysis of GbD27-6 in Gossypium barbadense

Tab.3 Secondary structure prediction of GbD27-6 protein

蛋白质折叠类型 Protein folding type	折叠个数 Number of folds	占比 Proportion(%)
无规则卷曲 Random coil	133	49.08
α-螺旋 α-spiral	88	32.47
延伸链 Extended chain	37	13.65
β-转角 β-corner	13	4.80

Fig.4 Tertiary structure prediction of GbD27-6 protein

Fig.5 Prediction of signal peptide of GbD27-6 in Gossypium barbadense

Fig.6 Prediction of transmembrane region of GbD27-6 in Gossypium barbadense

Fig.7 Conserved structural domain of GbD27-6 in Gossypium barbadense

Fig.8 Phosphorylation site of GbD27-6 in Gossypium barbadense

Fig.9 Predictive analysis of GbD27-6 and conservated motif in other plants

Fig.10 Analysis of amino acid sequence alignment between GbD27-6 and other plants

Fig.11 Phylogenetic tree analysis of GbD27-6 protein in sea island cotto

Fig.12 Expression of GbD27-6 gene of Gossypium barbadense in different tissues of floral organs Note:*,** and*** mean significant differences among different treatments of same gene at the 0.05, 0.01 and 0.001 probability levels,respectively,the same as below

Fig.13 Gene expression of GbD27-6 gene in different fiber development stages of Gossypium barbadense

References 18

[1]	孙洪影. 森林草莓FveD27基因分离鉴定及表达特性分析[D]. 沈阳: 沈阳农业大学, 2019.
	SUN Hongying. Isolation, identification and expression characteristics of FveD27 gene from forest strawberry[D]. Shenyang: Shenyang Agricultural University, 2019.
[2]	周晓燕. 独角金内酯的研究进展综述[J]. 安徽农学通报, 2016, 22(20):19-21.
	ZHOU Xiaoyan. Review of research progress of unicorn lactone[J]. Anhui Agricultural Science Bulletin, 2016, 22(20):19-21.
[3]	Kretzschmar T, Kohlen W, Sasse J, et al. A petunia ABC protein controls strigolactonedependent symbiotic signalling and branching[J]. Nature, 2012, 483(7389).
[4]	倪吉乐. REL2在水稻籽粒和分蘖发育中的作用机理研究[D]. 重庆: 西南大学, 2020.
	NI Jile. Study on the mechanism of REL2 in rice grain and tiller development[D]. Chongqing: Southwest University, 2020.
[5]	卓一南. 青天葵中独脚金内酯合成相关基因NfCCD7的克隆与表达载体的构建[D]. 广州: 广州中医药大学, 2018.
	ZHOU Yinan. Cloning and construction of expression vector of NfCCD7, a gene related toolactone synthesis in Sunflower[D]. Guangzhou: Guangzhou University of Traditional Chinese Medicine, 2018.
[6]	安奕霖, 尹克林. 独脚金内酯合成路径及抗逆性研究进展[J]. 现代农业科技, 2019,(9):120-122,125.
	An Yilin, YIN Kelin. Research progress on synthetic path and stress resistance of Lactoneof Lactone[J]. Modern Agricultural Science and Technology, 2019,(9):120-122,125.
[7]	Matusova R, Rani K, Verstappen F W A, et al. The strigolactone germination stimulantsof the plant- parasitic striga and orobanche spp. are derived from the carotenoid pathway[J]. Plant Physiology, 2005, 139(2):920-934. DOI URL
[8]	Santoro V, Schiavon M, Visentin I, et al. Strigolactones affect phosphorus acquisition strategies in tomato plants.[J]. Plant, Cell & Environment, 2021, 44(11).
[9]	Bunsick M, Toh S, Wong C, et al. SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga[J]. Nature Plants, 2020, 6(6):1-7. DOI
[10]	沈月, 陶宝杰, 华夏, 等. 独脚金内酯与激素互作调控根系生长的研究进展[J/OL]. 生物技术通报:1-8[2022-08-18].
	SHEN Yue, TAO Baojie, HUA Xia, et al. Advances in the study of root growth regulation by interactions of strigolactones and hormones[J/OL]. Biotechnology Bulletin:1-8[2022-08-18].
[11]	平文超, 张永江, 刘连涛, 等. 棉花根系生长分布及生理特性的研究进展[J]. 棉花学报, 2012, 24(2):183-190. DOI
	PING Wenchao, ZHANG Yongjiang, LIU Liantao, et al. Research progress on root growth distribution and physiological characteristics of cotton[J]. Cotton Science, 2012, 24(2):183-190.
[12]	Kun-Peng J, Lina B, Salim A B. From Carotenoids to Strigolactones[J]. Journal of Experimental Botany, 2017,(9):9.
[13]	唐超男. 外源独脚金内酯调控辣椒幼苗低温耐受性的生理与分子机制[D]. 兰州: 甘肃农业大学, 2021.
	TANG Chaonan. Physiological and molecular mechanism of exogenous bicephalolactone regulating low temperature tolerance of pepper seedlings[D]. Lanzhou: Gansu Agricultural University, 2021.
[14]	张艳培, 孙伟, 尹亮, 等. 水稻半矮化多分蘖突变体f2-132的表型分析和基因定位[J]. 植物遗传资源学报, 2014, 15(1):165-170.
	ZHANG Yanpei, SUN Wei, Yinliang, et al. Phenotypic analysis and gene mapping of rice semi-dwarf multi-tiller mutant f2-132[J]. Journal of Plant Genetic Resources, 2014, 15(1):165-170.
[15]	陈全家. 棉纤维发育相关基因转录组学、表达谱分析研究[D]. 北京: 中国农业大学, 2014.
	CHEN Quanjia. Analysis of transcriptomic expression profile of cotton fiber development related genes[D]. Beijing: China Agricultural University, 2014.
[16]	张荣祥, 杨清, 赵德刚. 新型植物激素—独脚金素内酯[J]. 生物学通报, 2011, 46(5):10-13.
	ZHANG Rongxiang, YANG Qing, ZHAO Degang. A New Phytohormone—strigolactones[J]. Biology Bulletin, 2011, 46(5):10-13.
[17]	苏维娜, 臧家富. 独脚金内酯生物学功能研究进展[J]. 山东农业科学, 2022, 54(5):159-164.
	SU Weina, ZANG Jiafu. Research progress on biological function of Lactone[J]. Shandong Agricultural Sciences, 2022, 54(5):159-164.
[18]	杜丽丽. 海岛棉枯萎病抗性遗传分析及抗病相关标记的评价[D]. 乌鲁木齐: 新疆农业大学, 2015.
	DU Lili. Genetic analysis of Fusarium wilt resistance of sea island cotton and evaluation of disease resistance related markers[D]. Urumqi: Xinjiang Agricultural University, 2015.

Cloning and bioinformatics analysis of β-carotene isomerase GbD27-6 gene in sea island cotton

海岛棉β-胡萝卜素异构酶GbD27-6基因克隆及表达分析

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 16

References 18

Related Articles 15

Recommended Articles

Metrics

[1]	ZHANG Chengjie, HU Haoran, DUAN Songjiang, WU Yifan, ZHANG Jusong. Effects of nitrogen-dense interaction on growth, development, yield and quality of Gossypium barbadense L. [J]. Xinjiang Agricultural Sciences, 2024, 61(8): 1821-1830.
[2]	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.
[3]	LU Guili, MIAO Shukui, WEI Jie, WEI Yurong, MI Xiaoyun, Hailiqiemu Maimaitiyiming. Structure and function analysis of spike protein of bovine coronavirus [J]. Xinjiang Agricultural Sciences, 2024, 61(11): 2844-2852.
[4]	HUANG Xinglei, WANG Weiran, WANG Meng, ZHU Jiahui, LIN Feng, QIN Guoli, YANG Jing, Alifu Aierxi, WU Quanzhong, KONG Jie. Diversity evaluation of machine-picked agronomic traits in Gossypium barbadense L. germplasm resources [J]. Xinjiang Agricultural Sciences, 2024, 61(1): 1-8.
[5]	JIN Juan, Subina Xiaokelaiti, Abudoukayoumu Ayimaiti, YANG Lei, HAO Qing, FAN Dingyu. Cloning and sequence analysis of expansin gene ZjEXPA8 in Huizao [J]. Xinjiang Agricultural Sciences, 2023, 60(9): 2223-2230.
[6]	GENG Feifei, MENG Chaomin, QING Guixia, ZHOU Jiamin, ZHANG Fuhou, LIU Fengju. Cloning and expression analysis of phosphorus efficient gene GhMYB4 in Gossypium hirsutum L. [J]. Xinjiang Agricultural Sciences, 2023, 60(6): 1406-1412.
[7]	Nasibai Abuduwahapu, GAO Xiaojuan, Kunduziayi Abudushalamu, LI Jiangwei. Cloning and Sequence Analysis of cDNA of C3d Gene of Bactrian Camel from Xinjiang [J]. Xinjiang Agricultural Sciences, 2023, 60(2): 493-500.
[8]	HU Wenran, ZHAO Zhun, SHAO Wukui, HUANG Quansheng. Identification of TCP family and analysis of tissue expression in upland cotton [J]. Xinjiang Agricultural Sciences, 2023, 60(11): 2627-2637.
[9]	Mayila Yusuyin, YANG Ni, XU Haijiang, YANG Yanlong, ZHANG Dawei, LI Chunping, SHI Bixian, LAI Chengxia. Bioinformatics and expression analysis of UGT gene family of cotton glycosyltransferase damaged by Helicoverpa armigera [J]. Xinjiang Agricultural Sciences, 2023, 60(10): 2396-2403.
[10]	CAI Yi, DAI Dongyang, TAN Hai, WANG Di, YANG Fen, WANG Ling, SHENG Yunyan. Analysis of Genetic Structure and Construction of Gene Editing Vector of Melon AMS Gene [J]. Xinjiang Agricultural Sciences, 2023, 60(1): 61-68.
[11]	LI Min, MA Ying, Huercha , HE Wenwen, SHI Qianyun, Alimujiang Jiapaer, JIANG Qian, Bayinchahan . Expression of Dm86 Gene of Dermacenter Marginatus and Analysis of Immunogenicity [J]. Xinjiang Agricultural Sciences, 2022, 59(9): 2324-2332.
[12]	DAI Qi, XU Ruiqiang, LI Ning, WANG Baike, WANG Juan, HUANG Shaoyong, Patiguli Aisimutuola, YU Qinghui. Genome-Wide Characterization and Analysis of GRF Gene Family in the Solanum pennellii [J]. Xinjiang Agricultural Sciences, 2022, 59(10): 2456-2465.
[13]	MIN Kaili, CHAO Xiangbao, TENG Lu, CAI Yongsheng, LEI Huichen, YAN Zhongjian, ZHENG Kai, CHEN Quanjia. Cloning and Expression Analysis of GbHCT10 Gene of Gossypium barbadense L. [J]. Xinjiang Agricultural Sciences, 2021, 58(2): 206-215.
[14]	WANG Degang, MA Guanghuang, HE Pengpeng, XIONG Renci, CAO Yu, ZHANG Ping, HAN Xu, YANG Minglu. Gene Sequences and Expression Analysis of Fatty Acid Δ9 Desaturase of Euzophera pyriella during Different Stages [J]. Xinjiang Agricultural Sciences, 2020, 57(5): 877-887.
[15]	MA Xingyu, JI Yujie, XUE Yuying, DU Mengfang, WEI Jizhen, YIN Xinming, LIU Xiaoguang, AN Shiheng. Gene Cloning and Molecular Characterization of Bursicon Gene in Helicoverpa armigera(Lepidoptera: Noctuidae) [J]. Xinjiang Agricultural Sciences, 2020, 57(4): 589-599.