陆地棉磷高效基因GhMYB4的克隆与表达分析

doi:10.6048/j.issn.1001-4330.2023.06.013

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (6): 1406-1412.DOI: 10.6048/j.issn.1001-4330.2023.06.013

• 种质资源·耕作栽培·生理生化·土壤肥料·分子遗传学 • 上一篇下一篇

陆地棉磷高效基因GhMYB4的克隆与表达分析

耿翡翡¹(), 孟超敏¹(), 卿桂霞¹, 周佳敏¹, 张富厚¹, 刘逢举²

1.河南科技大学农学院/洛阳市作物遗传改良与种质创新重点实验室,河南洛阳 471000
2.中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南安阳 455000

收稿日期:2022-10-27 出版日期:2023-06-20 发布日期:2023-06-20
通信作者: 孟超敏(1977-),男,陕西咸阳人,副教授,硕士生导师,研究方向为作物营养遗传育种,(E-mail) chaominm@haust.edu.cn
作者简介:耿翡翡(1997-),女,河南洛阳人,硕士研究生,研究方向为作物分子育种,(E-mail)2215293087@qq.com
基金资助:
国家自然科学基金项目“棉花根部低磷胁迫基因表达谱分析及相关基因的发掘”(31101184)

Cloning and expression analysis of phosphorus efficient gene GhMYB4 in Gossypium hirsutum L.

GENG Feifei¹(), MENG Chaomin¹(), QING Guixia¹, ZHOU Jiamin¹, ZHANG Fuhou¹, LIU Fengju²

1. College of Agriculture, Henan University of Science and Technology / Luoyang Key Laboratory of Crop Genetic Improvement and Germplasm Innovation, Luoyang Henan 471000, China
2. Institute of Cotton Research of CAAS/State Key Laboratory of Cotton Biology, Anyang Henan 455000, China

Received:2022-10-27 Online:2023-06-20 Published:2023-06-20
Correspondence author: MENG Chaomin(1977-), male, born in Xianyang, Shanxi, associate professor, master supervisor, mainly engaged in crop nutrition genetics and breeding research, (E-mail) chaominm@haust.edu.cn
Supported by:
National Natural Science Foundation of China "Analysis of Low Phosphorus Stress Gene Expression Profile and Related Genes in Cotton Root"(31101184)

摘要/Abstract

摘要：

【目的】基于陆地棉根部低磷胁迫基因谱芯片差异表达序列数据分析,挖掘相关基因,分析其克隆与表达,为研究棉花GhMYB4基因的生物学功能以及培育棉花磷高效利用新种质提供科学参考。【方法】克隆GhMYB4基因并进行基因组DNA与cDNA测序分析,运用生物信息学方法分析GhMYB4的基因结构和进化关系;采用半定量RT-PCR技术与荧光定量PCR(qRT-PCR)的方法检测该基因于根、茎、叶、花四个组织的基因表达量的变化。【结果】GhMYB4基因,开放阅读框序列长度774 bp,共编码257个氨基酸,属于MYB家族。半定量RT-PCR和qRT-PCR试验结果均表示,GhMYB4基因主要表达于根,中量表达于茎和叶,微量表达于花。【结论】获得GhMYB4基因及表达特性,该基因在棉花根中的表达量是最大的,在花中表达相对较小,中量表达于茎和叶。

关键词: 陆地棉; 低磷胁迫; 基因克隆; 表达分析; 生信分析

Abstract:

【Objective】 To excavate related genes based on the genome-wide expression profile of cotton seedlings under low phosphorus stress in the early stage and conducted analysis of their preliminary expression. This study results will provide a scientific reference for further research into the biological functions of GhMYB4 gene and the cultivation of new cotton germplasm with the efficient utilization of phosphorus.【Methods】 Genomic DNA and cDNA sequence of the gene were cloned and analyzed by bioinformatics method. Semi-quantitative RT-PCR and fluorescence quantitative PCR (QRT-PCR) were used to detect the changes of gene expression in root, stem, leaf and flower tissues. 【Results】 The GhMYB4 gene was cloned, whose open reading frame length was 774 bp. Altogether 257 amino acids were encoded and analysis revealed that this gene sequence belonged to the MYB family. The results of semi-quantitative RT-PCR and QRT-PCR showed that GhMYB4 gene was mainly expressed in root, medium expression in stems and leaf, and trace expression in flower. 【Conclusion】 The GhMYB4 gene is preliminarily obtained.The gene has the highest expression level in cotton roots,relatively less expressed in flowers,moderately expressed in stems and leaves.

Key words: Gossypium hirsutum L.; low phosphorus stress; gene cloning; expression analysis; bioinformatic analysis

中图分类号:

S562
S188

耿翡翡, 孟超敏, 卿桂霞, 周佳敏, 张富厚, 刘逢举. 陆地棉磷高效基因GhMYB4的克隆与表达分析[J]. 新疆农业科学, 2023, 60(6): 1406-1412.

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.

图/表 8

表1 所用引物信息

Tab.1 Primers used

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
Sense primer	TCGTTTCCGTTTCCCTACT
Anti-sense primer	CAATCATTTCATTTCCAAACTTCTG
M13F	TGTAAAACGACGGCCAGT
M13R	CAGGAAACAGCTATGACC
GhActin-F	ATCCTCCGTCTTGACCTTG
GhActin-R	TGTCCGTCAGGCAACTCAT
GhMYB4-F	TGGGAGTTAAAAACAGCAAACAATG
GhMYB4-R	CAATCATTTCATTTCCAAACTTCTG

图1 GhMYB4基因编码序列克隆注:M:Marker 2000;1:DNA的PCR扩增片段;2:cDNA的PCR扩增片段

Fig.1 Cloning of GhMYB4 CDS Note: M: Marker 2000; 1: PCR amplified fragment of DNA; 2: PCR amplified fragment of cDNA

图2. GhMYB4基因结构

Fig.2 GhMYB4 Gene structure

图3 GhMYB4与其他物种MYB家族同源蛋白的多序列比对

Fig.3 Multi-alignment of GhMYB4 amino acid sequence with other MYB family in different plants

图4 GhMYB4蛋白的三级结构预测

Fig.4 Predicted three-dimension structure of GhMYB4 protein

图5 GhMYB4基因系统进化树

Fig.5 GhMYB4 Phylogenetic tree

图6 半定量RT-PCR分析GhMYB4在棉花不同组织中的表达注:M:Marker 2000

Fig.6 Expression of GhMYB4 in different tissues of cotton by Semi-quantitative RT-PCR results Note:M:Marker 2000

图7 GhMYB4在棉花不同组织中的相对表达

Fig.7 Relative expression of GhMYB4 in different tissues of cotton

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陆地棉磷高效基因GhMYB4的克隆与表达分析

Cloning and expression analysis of phosphorus efficient gene GhMYB4 in Gossypium hirsutum L.

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