苦荞bHLH93转录因子响应铝胁迫的功能研究

doi:10.3864/j.issn.0578-1752.2024.16.003

摘要/Abstract

摘要：

【目的】荞麦是一种重要粮食和经济作物。与其他作物相比，荞麦具备较高的耐铝性。实验室前期研究，在铝离子胁迫下的苦荞转录组中发现一个可能与铝离子响应密切相关的转录因子FtbHLH93，探究FtbHLH93的功能，为解决土壤酸化引致铝毒害的问题及耐铝植物的选育提供思路与线索，并为荞麦耐铝性的分子机理解析奠定基础。【方法】以品苦1号的cDNA为模板克隆FtbHLH93，通过qRT-PCR检测其在苦荞不同组织和铝离子处理不同时间段的表达量；酵母系统鉴定转录激活活性；亚细胞定位确定其表达部位。检测过表达材料的黄酮类物质含量，在未处理和铝离子处理条件下测定SOD和POD活性。运用转录组分析差异表达基因，筛选潜在的下游靶基因，对其进行启动子预测，并利用双荧光素酶报告基因试验进行验证。【结果】FtbHLH93转录因子编码区长度为573 bp，编码190个氨基酸残基，预测蛋白分子量为21.759 kDa，等电点为8.64。qRT-PCR结果显示，FtbHLH93在苦荞根中表达量高，铝胁迫下基因表达定量分析表明，FtbHLH93在24 h时表达量最高。转录因子活性鉴定显示，FtbHLH93在酵母系统中无转录激活活性。亚细胞定位显示其定位在细胞核上。在铝离子处理下，过表达FtbHLH93植株的毛状根具有耐铝性，并且SOD和POD活性均显著高于对照组，过表达材料的黄酮类代谢物的检测结果显示，芦丁、儿茶素、烟花苷含量明显高于对照组。通过对转录组数据进行GO和KEGG富集分析，发现GO富集分析中，其与金属离子转运和镉锰离子条目有关，KEGG富集分析中，其与ABC转运蛋白有关，且挖掘出3个铝响应的候选下游靶基因，共表达分析发现其中2个候选下游靶基因与FtbHLH93的表达模式相似。【结论】FtbHLH93转录因子可能通过促进黄酮类物质的累积、SOD和POD活性的提高来缓解铝毒害，FtbHLH93可能靶向调控与铝响应有关的FtPinG0100930100.01、FtPinG0303102000.01和FtPinG0403996200.01。

关键词: 苦荞, 铝, bHLH, 过表达, 转录组, 双荧光素酶

Abstract:

【Objective】Buckwheat is an important cereal and economic crop. Compared with other crops, buckwheat has strong aluminum tolerance. A transcription factor FtbHLH93 in response to aluminum stress was identified in transcriptome data of aluminum treatment. Exploring the function of FtbHLH93 will provide ideas and clues for solving the problem of aluminum toxicity in acidic soil and molecular breeding of new varieties of buckwheat with aluminum tolerant, and provide theoretical basis for the molecular mechanism of tolerance aluminum in buckwheat.【Method】 The cDNA of Pinku1 was used as a template to clone FtbHLH93. qRT-PCR was used to detect the expression of FTbHLH93 in different tissues of Tartary buckwheat and at different time points after aluminum treatment. Yeast system was used to identify the transcriptional activation activity. The localization of intracellular expression was determined by subcellular localization. The flavonoid content of the overexpressed materials was examined, and SOD and POD activities were measured under untreated and Al-treated conditions. The differentially expressed genes were analyzed by transcriptome analysis, potential downstream target genes were screened, and their promoters were predicted. The dual luciferase reporter gene assay was used to verify the results.【Result】The coding region of FtbHLH93 transcription factor was 573 bp in length, encoding 190 amino acid residues. The predicted molecular weight of FtbHLH93 was 21.759 kDa, and its isoelectric point was 8.64. qRT-PCR results showed that FtbHLH93 was highly expressed in roots. The expression level of FtbHLH93 is highest at 24 h after aluminum treatment. FtbHLH93 is localized in the nucleus without self-activating activity. Overexpression of FtbHLH93 in Tartary buckwheat hairy roots enhanced aluminum tolerance, and the activities of SOD and POD were significantly higher than those of the control group. The detection results of flavonoid metabolites in the overexpressed FtbHLH93 hairy roots showed that the contents of rutin, catechin, and fireworks were significantly higher than those of the control group. GO enrichment analysis showed that it was related to metal ion transport and cadmium and manganese ion entries, and KEGG enrichment analysis showed that it was related to ABC transporter. Three genes responsive to aluminum stress may be downstream target genes of FtbHLH93, and co-expression analysis showed that two of the candidate downstream target genes had a similar expression pattern to FtbHLH93.【Conclusion】FtbHLH93 transcription factor may alleviate aluminum toxicity by promoting the accumulation of flavonoids and the increase of SOD and POD activities. FtbHLH93 may act as an upstream regulator to regulate the expression of FtPinG0100930100.01, FtPinG0303102000.01 and FtPinG0403996200.01.

Key words: tartary buckwheat, aluminum, bHLH, overexpression, transcriptome, Dual-luciferase

刘桐, 王志荣, 李伟, 刘洋, 王祥儒, 赖弟利, 何毓琦, 张凯旋, 赵振军, 周美亮. 苦荞bHLH93转录因子响应铝胁迫的功能研究[J]. 中国农业科学, 2024, 57(16): 3127-3141.

LIU Tong, WANG ZhiRong, LI Wei, LIU Yang, WANG XiangRu, LAI DiLi, HE YuQi, ZHANG KaiXuan, ZHAO ZhenJun, ZHOU MeiLiang. Function Analysis of bHLH93 Transcription Factor in Tartary Buckwheat in Response to Aluminum Stress[J]. Scientia Agricultura Sinica, 2024, 57(16): 3127-3141.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.16.003

https://www.chinaagrisci.com/CN/Y2024/V57/I16/3127

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基因ID Gene ID	表达水平Expression level	GO条目 GO term
FtPinG0707878600.01	下调 Down	铁离子结合，酸还原双加氧酶[需要铁]活性 Iron-binding, acid-reducing dioxygenase [iron required] activity
FtPinG0303292100.01	上调 Up	金属离子结合Metal ion binding
FtPinG0303287700.01	上调 Up	金属离子结合Metal ion binding
FtPinG0201816600.01	上调 Up	金属离子结合，负调控对缺水的反应 Metal ion binding and negative regulation of water deficiency response

基因ID Gene ID	表达水平Expression level	基因命名 Gene symbol	注释 Description	GO条目 GO term	表达集群 Express cluster	FC值 log₂(FoldChange)	P值 P-value
FtPinG0100930100.01	下调 Down	ABCB25	ABC转运蛋白B家族成员25 A member of the ABC transporter B family 25	铝离子跨膜转运活性Aluminium ion transmembrane transport activity	14	-1.045611368	0.002076521
FtPinG0303102000.01	上调 Up	ALMT4	铝活化苹果酸转运蛋白4 Aluminum activates malate transporter 4	离子通道活性 Ion channel activity	13	2.282465572	2.69E-10
FtPinG0403996200.01	上调 Up	PLDGAMMA1	磷脂酶Dγ1 Phospholipase Dγ1	响应铝离子，钙离子结合 In response to aluminum ions, calcium ions binding	13	1.168803299	3.45E-08