海岛棉β-胡萝卜素异构酶GbD27-6基因克隆及表达分析

doi:10.6048/j.issn.1001-4330.2023.12.002

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (12): 2869-2877.DOI: 10.6048/j.issn.1001-4330.2023.12.002

• 作物遗传育种·种质资源·分子遗传学·土壤肥料 • 上一篇下一篇

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

收稿日期:2023-04-09 出版日期:2023-12-20 发布日期:2024-01-03
通信作者: 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)

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)

摘要/Abstract

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

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

中图分类号:

S562

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.

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.

图/表 16

表1 引物序列

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

表2 生信分析软件

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

图1 海岛棉GbD27-6基因凝胶电泳图注:M:DL2000 DNA maker;1:GbD27-6基因的扩增产物

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

图2 海岛棉GbD27-6基因菌液凝胶电泳图注:M:DL2000 DNA maker;2:GbD27-6基因菌液的阳性鉴定

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

图3 海岛棉GbD27-6的亲疏水性

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

表3 GbD27-6蛋白二级结构预测

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

图4 GbD27-6蛋白三级结构预测

Fig.4 Tertiary structure prediction of GbD27-6 protein

图5 海岛棉GbD27-6的信号肽预测

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

图6 海岛棉GbD27-6的跨膜区预测

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

图7 海岛棉GbD27-6的保守结构域

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

图8 海岛棉GbD27-6的磷酸化位点

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

图9 GbD27-6与其他植物保守基序预测分析

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

图10 GbD27-6与其他植物氨基酸序列比对

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

图11 海岛棉GbD27-6蛋白系统进化树

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

图12 海岛棉GbD27-6基因在花器官不同组织中的表达量注:*、**和***表示同一基因不同处理间在0.05、0.01、0.001水平差异显著,下同

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

图13 GbD27-6基因在海岛棉不同纤维发育时期的基因表达量

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

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海岛棉β-胡萝卜素异构酶GbD27-6基因克隆及表达分析

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

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