盐胁迫下辣椒CaPIF4的表达特性与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1045

生物技术通报 ›› 2024, Vol. 40 ›› Issue (4): 148-158.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1045

盐胁迫下辣椒CaPIF4的表达特性与功能分析

李慧¹(), 文钰芳¹, 王悦¹, 纪超¹, 石国优¹, 罗英², 周勇¹, 李志敏¹, 吴晓玉¹, 杨有新²(), 刘建萍¹()

1.江西农业大学生物科学与工程学院江西省农业微生物资源开发与利用工程实验室，南昌 330045
2.江西农业大学农学院江西省果蔬保鲜与无损检测重点实验室，南昌 330045

收稿日期:2023-11-07 出版日期:2024-04-26 发布日期:2024-04-30
通讯作者: 刘建萍，女，博士，副教授，研究方向：植物分子生物学；E-mail: JianpingLiu@jxau.edu.cn；
杨有新，男，博士，教授，研究方向：设施园艺；E-mail: yangyouxin@jxau.edu.cn
作者简介:李慧，女，硕士研究生，研究方向：植物分子生物学；E-mail: lh15736630394@163.com
第一联系人：
文钰芳为本文共同第一作者
基金资助:
江西省主要学科学术和技术带头人培养计划，青年人才项目(20204BCJL23044);国家自然科学基金项目(32060047);江西省自然科学基金面上项目(20232BAB205041);赣鄱俊才支持计划，高层次和急需紧缺海外人才引进项目(20232BCJ25068)

Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress

LI Hui¹(), WEN Yu-fang¹, WANG Yue¹, JI Chao¹, SHI Guo-you¹, LUO Ying², ZHOU Yong¹, LI Zhi-min¹, WU Xiao-yu¹, YANG You-xin²(), LIU Jian-ping¹()

1. College of Bioscience and Bioengineering, Jiangxi Agricultural University, Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, Nanchang 330045
2. College of Agronomy, Jiangxi Agricultural University, Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables, Nanchang 330045

Received:2023-11-07 Published:2024-04-26 Online:2024-04-30

摘要/Abstract

摘要：

【目的】研究辣椒光敏色素互作因子4（phytochrome-interacting factor 4, PIF4）响应盐胁迫的分子机制，为探究辣椒响应盐胁迫的分子机理和选育耐盐品种提供理论基础。【方法】以‘Zunla-1’cDNA为模板克隆CaPIF4，利用生物信息软件分析该基因所编码蛋白的理化性质，实时荧光定量PCR（RT-qPCR）技术和病毒诱导基因沉默（virus induced gene silencing, VIGS）技术研究盐胁迫处理后CaPIF4表达模式及在盐胁迫中的作用。【结果】CaPIF4在辣椒根、茎、叶、花和果实各组织均有表达，但在叶片中表达水平最高；随着300 mmol/L NaCl处理时间的增加，CaPIF4表达量升高，其中，在处理2 h后表达量达到最高；利用VIGS技术沉默CaPIF4，将对照组（CK）和基因瞬时沉默组（pTRV-CaPIF4）进行300 mmol/L NaCl处理后发现，基因沉默组植株与空载组（CK）相比，萎蔫程度更为严重，说明基因瞬时沉默组（pTRV-CaPIF4）植株耐盐性降低；亚细胞定位结果显示，CaPIF4定位于细胞核；酵母转录活性分析发现CaPIF4具有转录激活活性。高盐胁迫下，辣椒叶片过氧化氢含量升高，过氧化物酶活性升高。染色结果显示，沉默CaPIF4后，导致H₂O₂和O^2-含量显著升高。【结论】CaPIF4作为转录因子可能参与调控辣椒的盐耐受性。

关键词: 辣椒, 盐胁迫, CaPIF4, 转录因子, 活性氧

Abstract:

【Objective】Studying the molecular mechanisms of the pepper photoreceptor pigment phytochrome-interacting factor 4（PIF4）in response to salt stress plays an important role in understanding the molecular mechanisms of pepper's response to salt stress and breeding salt-tolerant varieties. 【Method】Using ‘Zunla-1’ cDNA as a template, CaPIF4 was cloned. Then the physical and chemical properties of CaPIF4 were analyzed using bioinformatics software. And the expression pattern and salt stress-induced expression was studied by real-time fluorescence quantitative PCR（RT-qPCR）and virus-induced gene silencing technology（VIGS）. 【Result】CaPIF4 was expressed in various tissues of pepper, including roots, stems, leaves, flowers, and fruits, with the highest expression in the leaves. And the expression of CaPIF4 increased with the extension of 300 mmol/L NaCl treatment, and reached the highest at 2 h after treatment. CaPIF4 gene was silenced using VIGS technology. The control group（CK）and gene transient silencing group（pTRV-CaPIF4）were treated in 300 mmol/L NaCl. And the phenotype showed that pTRV-CaPIF4 plants after salt stress presented more obvious salty damage compared with CK. Using VIGS technology to silence CaPIF4, the CK and the pTRV-CaPIF4 were treated with 300 mmol/L NaCl. It was found that the plants in the pTRV-CaPIF4 group were more severely wilted compared to CK after gene silencing, indicating a decrease in salt tolerance in the pTRV-CaPIF4 group. Subcellular localization results showed that CaPIF4 was located in the nucleus. Yeast transcriptional activity analysis revealed that CaPIF4 had transcriptional activation activity. Under high salt stress, the hydrogen peroxide content and antioxidant enzyme activity in the pepper leaves increased. Staining results showed that silencing the CaPIF4 gene resulted in a significant increase in H₂O₂ and O^2- levels. 【Conclusion】CaPIF4, as a transcription factor, may be involved in regulating salt tolerance in peppers.

Key words: Capsicum annuum, salt stress, CaPIF4, transcription factor, ROS

李慧, 文钰芳, 王悦, 纪超, 石国优, 罗英, 周勇, 李志敏, 吴晓玉, 杨有新, 刘建萍. 盐胁迫下辣椒CaPIF4的表达特性与功能分析[J]. 生物技术通报, 2024, 40(4): 148-158.

LI Hui, WEN Yu-fang, WANG Yue, JI Chao, SHI Guo-you, LUO Ying, ZHOU Yong, LI Zhi-min, WU Xiao-yu, YANG You-xin, LIU Jian-ping. Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress[J]. Biotechnology Bulletin, 2024, 40(4): 148-158.

图/表 12

表1 引物列表

Table1 List of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
CaPIF4-F	ATGAATCCTTGTCTTCCTG
CaPIF4-R	TCAAACCTGAGAAGCACC
CaPIF4-qPCR-F	TGGTTGATCCATCAATCCCTT
CaPIF4-qPCR-R	CAAACCTGAGAAGCACCCTGA
CaPIF4-VIGS-F	TGAGTAAGGTTACCGAATTCATGAATCCTTGTC- TTCCTGAAT
CaPIF4-VIGS-R	GTGAGCTCGGTACCGGATCCACAAAATACTTTG- TCAAATGAAT
pGBKT7-CaPIF4-F	ATGGCCATGGAGGCCGAATTCATGAATCCTTGT- CTTCCTGAATGG
pGBKT7-CaPIF4-R	CCGCTGCAGGTCGACGGATCCAACCTGAGAAGCACCCTG
p3301-CaPIF4-F	ACGGGGGACTCTTGACCATGGATGAATCCTTG- TCTTCCTGAATGG
p3301-CaPIF4-F	GCCCTTGCTCACCATCCATGGAACCTGAGAAGCACCCTG
CaACTIN-F	AGGGATGGGTCAAAAGGATGC
CaACTIN-F	GAGACAACACCGCCTGAATAGC

图1 CaPIF4的扩增 M：BM2000 DNA marker；1-2：2个重复

Fig. 1 PCR amplification of CaPIF4 M: BM2000 DNA marker. 1-2: Two repeats

图2 CaPIF4的序列分析 A：CaPIF4的亲水性分析；B：CaPIF4保守结构域分析；C：CaPIF4二级结构；D：CaPIF4三级结构

Fig. 2 Sequence analysis of CaPIF4 A: Hydrophilicity analysis of CaPIF4 protein. B: Conserved domain analysis of CaPIF4. C: Secondary structure of CaPIF4 protein. D: Tertiary structure of CaPIF4 protein

图3 CaPIF4的表达模式分析 A：不同组织中CaPIF4的表达分析；B：盐胁迫下CaPIF4在叶中的表达分析。平均值和标准差分别由3个独立实验得到，每个实验3株植物。不同小写字母表示差异有统计学意义，P≤0.05

Fig. 3 Expression pattern analysis of CaPIF4 A: Expression analysis of CaPIF4 in different tissues. B: Expression analysis of CaPIF4 in the leaves under salt stress. Mean and SD values were obtained from three independent experiments, with 3 plants per experiments. The different letters indicate significant differences at P ≤0.05

图4 pTRV2-CaPIF4载体的构建 A：pTRV2-CaPIF4载体的酶切验证；B：pTRV2-CaPIF4载体序列比对；M：BM200 DNA marker；1-3：3个重复

Fig. 4 Construction of pTRV2-CaPIF4 vector A: Enzyme digesting verification of pTRV2-CaPIF4 vector; B: pTRV2-CaPIF4 vector sequence alignment; M: BM2000 DNA marker; 1-3: three repeats

图5 VIGS沉默辣椒植株表型

Fig. 5 Phenotypic observation of pepper plants silenced by VIGS

图6 CaPIF4沉默效率检测图中误差线表示标准偏差，星号代表显著差异（*P<0.05，** P<0.01，***P<0.001），下同

Fig. 6 Detection of CaPIF4 gene silencing efficiency The error line in the figure refers to the standard deviation. * indicate statistical significance （*P<0.05，**P<0.01，***P<0.001 ） compared to control. The same below

图7 CaPIF4-GFP融合蛋白亚细胞定位

Fig. 7 Subcellular localization of CaPIF4-GFP fusion protein

图8 CaPIF转录自激活活性分析

Fig. 8 Transcription self-activation activity analyses of CaPIF4

图9 盐处理下CaPIF4沉默植株表型分析

Fig. 9 Phenotype analysis of CaPIF4 gene silencing plants under salt stress

图10 盐胁迫前后对照组和VIGS沉默植株第3、4叶片中ROS积累情况 A：3,3' -二氨基联苯胺（DAB）染色；B：氮蓝四唑（NBT）染色

Fig. 10 ROS accumulation in the control group and VIGS silenced plants in the 3rd and 4th leaves before and after salt stress A: 3,3'-Diaminobenzidine (DAB) staining. B: Nitro-blue tetrazolium (NBT) staining

图11 盐胁迫下辣椒植株的生理生化指标测定

Fig. 11 Determination of physiological and biochemical indexes of pepper plants under salt stress

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盐胁迫下辣椒CaPIF4的表达特性与功能分析

Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress

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