烟草叶片响应镉胁迫的差异表达基因鉴定及分析

doi:10.3724/SP.J.1006.2024.34141

作物学报 ›› 2024, Vol. 50 ›› Issue (4): 944-956.doi: 10.3724/SP.J.1006.2024.34141

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

烟草叶片响应镉胁迫的差异表达基因鉴定及分析

张慧(), 张欣雨, 袁旭, 陈伟达, 杨婷()

江汉大学生命科学学院 / 汉江流域特色生物资源保护开发与利用工程技术研究中心, 湖北武汉 430056

收稿日期:2023-08-15 接受日期:2023-10-23 出版日期:2024-04-12 网络出版日期:2023-11-15
通讯作者: * 杨婷, E-mail: yangtingYT2016@163.com
作者简介:E-mail: zhanghui313355@outlook.com
基金资助:
湖北省自然科学基金计划项目(2022CFB909);国家自然科学基金青年项目(31900095);江汉大学一流学科建设重大专项计划项目(2023XKZ016)

Transcriptome analysis of tobacco in response to cadmium stress

ZHANG Hui(), ZHANG Xin-Yu, YUAN Xu, CHEN Wei-Da, YANG Ting()

College of Life Sciences, Jianghan University / Hubei Engineering Research Center for Protection and Utilization of Special Biological Resources in the Hanjiang River Basin, Wuhan 430056, Hubei, China

Received:2023-08-15 Accepted:2023-10-23 Published:2024-04-12 Published online:2023-11-15
Contact: * E-mail: yangtingYT2016@163.com
Supported by:
Natural Science Foundation of Hubei Province of China(2022CFB909);Youth Fund Project of the National Natural Science Foundation of China(31900095);First-Class Discipline Construction and Special Program of Jianghan University(2023XKZ016)

摘要/Abstract

摘要：

烟草具有超富集镉的能力, 严重降低烟叶品质, 影响其经济价值。为了阐释烟草响应镉胁迫的分子机制, 本研究采集了镉浓度为0和500 μmol L^-1培养条件下的烟草叶片进行转录组测序。共获得76.94 Gb有效数据(Clean data), Q30 碱基百分比均达到95.43%以上; 在镉胁迫的烟草叶片中, 共筛选出7735个差异表达基因, 其中4833个基因表达上调, 2902个基因表达下调, 并通过qRT-PCR分析验证了转录组数据的可靠性。对差异转录本进行GO和KEGG富集分析, GO注释表明差异基因涉及代谢过程、应激反应、细胞结构体、催化活性和转录调节活性等; KEGG富集分析表明上调差异基因主要富集在氨基酸的生物合成、碳代谢、氧化磷酸化和柠檬酸循环等通路, 下调差异基因则主要富集在光合作用、次生代谢产物的生物合成、代谢途径和植物激素信号转导途径。进一步分析植物激素信号转导通路发现, 共有8条植物激素途径以不同的表达方式参与烟草对镉胁迫的响应。激素喷施烟草的实验结果表明, 叶片通过调控赤霉素、油菜素内酯和茉莉酸途径以应对镉胁迫; 拟南芥激素信号缺失突变体验证实验表明赤霉素、油菜素内酯、茉莉酸和乙烯途径均响应镉胁迫。综上所述, 本文以转录组分析探究了烟草叶片响应镉胁迫的调控网络, 以期为提高作物抗逆性的遗传改良提供理论依据。

关键词: 普通烟, 镉胁迫, 转录组分析, 调控机制, 植物激素

Abstract:

With the development of industrialization process in society, the problem of cadmium (Cd) pollution in soil is increasing. However, Nicotiana tabacum has a strong Cd enrichment capacity in leaves, which seriously affects its economic value. To investigate the mechanism by which tobacco responds to Cd stress, tobacco leaves were harvested from the culture solution with Cd concentrations of 0 μmol L^-1 and 500 μmol L^-1 for subsequent transcriptome sequencing. In this study, a total of 76.94 Gb clean data was obtained, with Q30 base percentage exceeding 95.43%. The results showed that 7735 differentially expressed genes (DEGs) were screened under Cd stress conditions, including 4833 up-regulated genes and 2902 down-regulated genes. The reliability of transcriptome data was verified by qRT-PCR analysis to detect the expression patterns of candidate gene. Gene ontology (GO) annotation as well as Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed on differentially expressed transcripts. GO functional enrichment revealed that the differentially expressed genes were mainly distributed in metabolic processes, response to stimulus, cellular anatomical entity, catalytic activity, and transcription regulator activity. Meanwhile, KEGG analysis showed that the up-regulated differentially expressed genes were mainly involved in biosynthesis of amino acids, carbon metabolism, oxidative phosphorylation, and citrate cycle. Down-regulated differentially expressed genes were primarily enriched in photosynthesis, biosynthesis of secondary metabolites, metabolic pathways, and plant hormone signal transduction. Further analysis of plant hormone signal transduction pathways revealed that there were eight plant hormone pathways involved in response to cadmium stress in tobacco, and the relative expression patterns of different hormone gene member were also different. Experimental results from plant hormone application on tobacco leaves demonstrated that the regulation of gibberellins, brassinosteroids, and jasmonic acid pathways played roles in tobacco’s response to cadmium stress. The experimental results of Arabidopsis hormone signal mutant showed that plants respond to cadmium stress by regulating ethylene, gibberellin, brassinosteroid, and jasmonic acid pathways. In conclusion, this study not only explores the regulatory network of tobacco resistance to Cd stress, but also lays a theoretical foundation for the genetic improvement of crop resistance.

Key words: Nicotiana tabacum, cadmium stress, transcriptome analysis, regulatory mechanism, plant hormone

张慧, 张欣雨, 袁旭, 陈伟达, 杨婷. 烟草叶片响应镉胁迫的差异表达基因鉴定及分析[J]. 作物学报, 2024, 50(4): 944-956.

ZHANG Hui, ZHANG Xin-Yu, YUAN Xu, CHEN Wei-Da, YANG Ting. Transcriptome analysis of tobacco in response to cadmium stress[J]. Acta Agronomica Sinica, 2024, 50(4): 944-956.

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基因编号 Gene ID	正向引物序列 Forward sequences (5°-3')	反向引物序列 Reverse sequences (5°-3')
NtEF1α	GCTGTAACAAGATGGATGC	AGATGGGGACAAAGGGGAT
Nitab4.5_0001799g0060	TGATCGAACGGACCATTAT	AAGCCTTATTCCAACTCTG
Nitab4.5_0000137g0100	GATCTTCTGAGTACCCGTAT	CAACTTGATTCCTCCTTC
Nitab4.5_0001834g0020	GTAGCTGCATTGGCCCTTAG	CTGTTGCTGCCTTCATTTCC
Nitab4.5_0001603g0110	ATAGTCAACAACTCCCTC	CCTTCCAGCAGAATTAGA
Nitab4.5_0002739g0090	GTTGTTGTCGGAACTATGT	ACCAGGCTTCTGATAAACT
Nitab4.5_0000303g0130	TCTCAGTCTCCGTTCCTCTA	CTTTAAGCCTCTGGGTGTAG
Nitab4.5_0000786g0090	TCAACCTTTCTCGCTTCTGC	CTGACCTCCCAAATTGCCTA

样品 Sample	原始读长 Raw reads	有效读长 Clean reads	有效碱基 Clean bases	GC含量 GC content (%)	Q30%
Cd-1	117,197,630	114,117,568	16.28	51.24	96.27
Cd-2	92,057,888	89,923,200	12.87	52.87	95.43
Cd-3	92,277,960	89,807,396	12.79	52.84	96.01
CK-1	80,695,074	79,549,980	11.55	52.49	95.62
CK-2	90,502,566	88,261,644	12.36	53.06	96.45
CK-3	78,412,514	77,125,276	11.09	52.56	96.25

基因编号 Gene ID	倍数变化 log₂ (Fold Change)	校正后P值 P_adj-value	显著性 Significant	相关功能 Related functions
Nitab4.5_0001799g0060	6.01645779051992	5.58600524883223E-53	上调Up	NADH氧化酶 NADHoxidase
Nitab4.5_0000137g0100	4.93283684860978	5.82620770685452E-36	上调Up	脂氧合酶 Lipoxygenase
Nitab4.5_0001834g0020	2.2757421379006	0.00106352851309622	上调Up	AP2/ERF结构域 AP2/ERFdomain
Nitab4.5_0001603g0110	2.27445197002978	5.39888738311809E-07	上调Up	WRKY转录因子 DNA-bindingWRKY
Nitab4.5_0002739g0090	-2.11819490857942	3.37346132804127E-19	下调Down	光系统天线蛋白Photosystemantennaprotein-like
Nitab4.5_0000303g0130	-1.68510291076442	2.83044174648409E-15	下调Down	核糖体蛋白L21 RibosomalproteinL21
Nitab4.5_0000786g0090	-2.10064535366169	1.06320970799878E-18	下调Down	植物抗坏血酸过氧化物酶Plantascorbateperoxidase

基因编号 Gene ID	倍数变化 log₂ (Fold Change)	校正后P值 P_adj-value	显著性 Significant	基因符号 Gene symbol
Nitab4.5_0001331g0110	2.29205916646639	0.0000160056470884177	上调Up	ABCC10
Nitab4.5_0001898g0080	2.22733652858828	0.000386104024008464	上调Up	ABCC15
Nitab4.5_0000106g0390	1.31870696593661	0.0212689692966056	上调Up	ABCG3
Nitab4.5_0000496g0120	-2.59158544291489	1.60564591983942E-08	下调Down	ABCG5
Nitab4.5_0006252g0030	1.31746228592749	0.0341152515360627	上调Up	ABCG20
Nitab4.5_0000221g0040	2.28821296882979	0.00370517654747173	上调Up	ABCG21
Nitab4.5_0002381g0060	10.210671343785	0.0000908617814568147	上调Up	CAX18
Nitab4.5_0002478g0050	2.03423844364883	2.69008075629427E-06	上调Up	HMA1
Nitab4.5_0003524g0040	2.10793204476971	0.0000026522303535919	上调Up	HMA3
Nitab4.5_0000492g0070	2.09307506718162	0.000256620985201009	上调Up	HMA5
Nitab4.5_0000267g0020	-1.05182186076751	0.0000603639575690097	下调Down	HMA32
Nitab4.5_0000915g0200	1.48223660266316	0.0032272780694283	上调Up	HMA39
Nitab4.5_0001119g0080	2.64624606789856	7.33048509501148E-08	上调Up	NRAMP 3
Nitab4.5_0004147g0050	5.31130934808622	9.33236566781447E-10	上调Up	NRAMP 6
Nitab4.5_0004048g0010	3.17293738846753	2.66390391106025E-12	上调Up	PT2.11
Nitab4.5_0000081g0170	-1.37360555768205	7.90967873459083E-09	下调Down	PT4.5
Nitab4.5_0000417g0150	2.1229862013314	0.00122100988376109	上调Up	PT4.6
Nitab4.5_0003449g0030	1.30297458667808	0.011869363195499	上调Up	PT6.4
Nitab4.5_0000166g0100	7.21539127135386	1.40896298333894E-45	上调Up	PT7.3

烟草叶片响应镉胁迫的差异表达基因鉴定及分析

Transcriptome analysis of tobacco in response to cadmium stress

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