水稻响应盐胁迫关键转录因子的鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-0802

摘要/Abstract

摘要：

盐胁迫是影响水稻生产的重要非生物胁迫之一。本研究以盐胁迫条件下的水稻转录组数据为材料,通过HTSeq和DESeq软件分析转录因子在转录组水平上的表达变化。主要结果如下：水稻在盐胁迫1、3和6 h三个时间点共有26个相同的差异表达转录因子,通过蛋白互作筛选出14个基因组成的重要模块;重要模块基因GO分析主要富集在ATP结合功能、对胁迫反应的过程以及细胞质膜囊泡组分上,KEGG分析主要富集在内质网蛋白质加工和内吞作用的通路上;同时鉴定出受盐胁迫诱导表达的关键热激转录因子基因Os03g0745000和Os06g0553100。推测这两个基因可能通过调控HSP70基因的转录表达,影响蛋白质折叠和组装,从而在水稻响应盐胁迫的过程中发挥作用。该研究为后续进行水稻遗传改良提供了候选基因,对进一步认识耐盐机制提供科学启示。

关键词: 水稻, 关键转录因子, 盐胁迫, 蛋白互作, 转录组

Abstract:

Salt stress is one of the most important abiotic stresses affecting rice production. In this study,the transcriptome data of rice under salt stress were used to analyze the expression changes of transcription factors at the transcriptome level by HTSeq and DESeq software. The main results were as follows：there were 26 identical transcription factors differentially expressed in rice at 1,3 and 6 h of salt stress,and an important module containing 14 genes was screened by protein interaction. GO analysis of important modules showed that 14 genes were mainly enriched in ATP binding,stress response and cytoplasmic membrane-bounded vesicle. KEGG analysis showed that it was mainly enriched in endoplasmic reticulum protein processing and endocytosis pathway. Meanwhile,the key heat shock transcription factor gene Os03g074500 and Os06g0553100 induced by salt stress were identified. It is speculated that these two genes may play a significant role in the process of rice salt stress by regulating the transcription and expression of HSP70 gene and affecting protein folding and assembly. This study provides candidate genes for subsequent genetic improvement of rice and provides scientific enlightenment for further understanding of salt tolerance mechanism.

Key words: rice, key transcription factors, salt stress, protein interaction, transcriptome

张斌, 杨昕霞. 水稻响应盐胁迫关键转录因子的鉴定[J]. 生物技术通报, 2022, 38(3): 9-15.

ZHANG Bin, YANG Xin-xia. Identification of Key Transcription Factors in Response to Salt Stress in Rice[J]. Biotechnology Bulletin, 2022, 38(3): 9-15.

图/表 8

表1 qRT-PCR反应引物序列和基因注释信息

Table 1 Primer sequence for qRT-PCR and gene annotation information

基因IDGene ID	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）	注释Description
Os03g0322900	CCAAGAACAAGCTGGGCGAG	CTTGAACTCCGTCGCCTTCT	OsLEA3-2^[6]
Os11g0454300	GCTCCAGCTCAAGCTCGTC	CATGGCATGCTGCTGCTC	RAB21^[7]
Os03g0745000	CATTGCGCGTCATCGTGAAA	GACCTCGTACGTCTTGCACA	OsHsfA2a
Os06g0553100	CCGTTCGTGTGGAAGACGTA	ACCATAGGTGTTGAGCTGGC	OsHsfC2b
Os10g0510000	AGCTATCGTCCACAGGAA	ACCGGAGCTAATCAGAGT	OsActin
Os03g0277300	ACCATCAAGAACGCCGTCAT	TGAGGACATTCTTCTCGCCG	OsHSP70
Os03g0276500	ATGATCGGCGTGCAGTACAA	GACGGTGCTTCCGAGATAGG	OsHSP71.1

图1 测序样品关系 A：样品间PCA分析图;B：样品相关性热图。0 h、1 h、3 h和6 h表示0 h、1 h、3 h和6 h取样时间点,-1、-2和-3表示3个重复

Fig. 1 Relationship among sequencing samples A：PCA analysis diagram of samples. B：Heat map of sample correlation. 0,1,3 and 6 h represent 0,1,3 and 6 h sampling time points. -1,-2 and - 3 represent three repetitions

图2 盐胁迫差异表达基因和相同差异表达基因筛选图 A：盐胁迫1 h差异表达基因火山图;B：盐胁迫3 h差异表达基因火山图;C：盐胁迫6 h差异表达基因火山图;D：响应盐胁迫差异表达基因韦恩图

Fig. 2 Screening of differentially expressed genes(DEGs) and identifical DEGs under salt stress A：Differentially expressed genes under salt stress for 1 h. B：Volcano diagram of differentially expressed genes under salt stress for 3 h. C：Volcano diagram of differentially expressed genes under salt stress for 6 h. D：Venn diagram of differentially expressed genes in response to salt stress

表2 26个相同差异表达TF基因在STRING中的注释

Table 2 Notes of 26 identical differentially expressed TF genes in STRING

TF基因TF Gene	STRING中名称STRING ID	注释Annotation
Os11g0151002	OS11T0151002-00	Os11g0151002蛋白质 Os11g0151002 protein
Os06g0553100	OS06T0553100-01	热应激转录因子C-2b Heat stress transcription factor C-2b
Os04g0398000	OS04T0398000-02	Os04g0398000蛋白 Os04g0398000 protein
Os03g0820300	OsJ_13136	C2H2转录因子 C2H2 transcription factor
Os09g0522100	DREB1H	介导高盐和脱水诱导的转录 Mediates high salinity- and dehydration-inducible transcription
Os07g0688200	OS07T0688200-01	MYB转录因子 MYB transcription factor
Os03g0745000	Hsf4	热应激转录因子A-2a Heat stress transcription factor A-2a
Os08g0200600	OS08T0200600-01	Os08g0200600蛋白质 Os08g0200600 protein
Os05g0437100	OS05T0437100-00	Os05g0437133蛋白质 Os05g0437133 protein
Os12g0564100	OS12T0564100-01	无注释 Annotation not available
Os06g0670300	P0485A07.2	Os06g0670300蛋白 Os06g0670300 protein
Os02g0638650	P0010C01.6	含AP2结构域的类转录因子 AP2 domain-containing transcription factor-like
Os01g0693400	OS01T0693400-01	含AP2/ERF和B3结构域的蛋白质 AP2/ERF and B3 domain-containing protein
Os11g0168500	OS11T0168500-00	无注释 Annotation not available
Os01g0859100	OS01T0859100-01	Os01g0859100蛋白 Os01g0859100 protein
Os01g0318400	OS01T0318400-00	Os01g0318400蛋白质 Os01g0318400 protein
Os09g0522200	ERF24	脱水反应元件结合蛋白1A Dehydration-responsive element-binding protein 1A
Os03g0820400	ZFP37	C2H2型锌指转录因子ZFP37 C2H2 type zinc finger transcription factor ZFP37
Os10g0571600	OS10T0571600-01	Os10g0571600蛋白质 Os10g0571600 protein
Os01g0626400	WRKY11	WRKY转录因子11 WRKY transcription factor 11
Os03g0838800	OS03T0838800-00	锌指蛋白 Zinc finger protein
Os05g0132700	OS05T0132700-01	Os05g0132700蛋白质 Os05g0132700 protein
Os03g0287400	OsJ_10424	LOB结构域蛋白1 LOB domain protein 1
Os04g0529100	OS04T0529100-01	Os04g0529100蛋白质 Os04g0529100 protein
Os11g0154900	OS11T0154900-01	BZIP转录因子家族蛋白 BZIP transcription factor family protein
Os01g0298400	OsJ_01424	P型r2r3MYB蛋白 P-type r2r3 MYB protein

表2 26个相同差异表达TF基因在STRING中的注释

Table 2 Notes of 26 identical differentially expressed TF genes in STRING

TF基因TF Gene	STRING中名称STRING ID	注释Annotation
Os11g0151002	OS11T0151002-00	Os11g0151002蛋白质 Os11g0151002 protein
Os06g0553100	OS06T0553100-01	热应激转录因子C-2b Heat stress transcription factor C-2b
Os04g0398000	OS04T0398000-02	Os04g0398000蛋白 Os04g0398000 protein
Os03g0820300	OsJ_13136	C2H2转录因子 C2H2 transcription factor
Os09g0522100	DREB1H	介导高盐和脱水诱导的转录 Mediates high salinity- and dehydration-inducible transcription
Os07g0688200	OS07T0688200-01	MYB转录因子 MYB transcription factor
Os03g0745000	Hsf4	热应激转录因子A-2a Heat stress transcription factor A-2a
Os08g0200600	OS08T0200600-01	Os08g0200600蛋白质 Os08g0200600 protein
Os05g0437100	OS05T0437100-00	Os05g0437133蛋白质 Os05g0437133 protein
Os12g0564100	OS12T0564100-01	无注释 Annotation not available
Os06g0670300	P0485A07.2	Os06g0670300蛋白 Os06g0670300 protein
Os02g0638650	P0010C01.6	含AP2结构域的类转录因子 AP2 domain-containing transcription factor-like
Os01g0693400	OS01T0693400-01	含AP2/ERF和B3结构域的蛋白质 AP2/ERF and B3 domain-containing protein
Os11g0168500	OS11T0168500-00	无注释 Annotation not available
Os01g0859100	OS01T0859100-01	Os01g0859100蛋白 Os01g0859100 protein
Os01g0318400	OS01T0318400-00	Os01g0318400蛋白质 Os01g0318400 protein
Os09g0522200	ERF24	脱水反应元件结合蛋白1A Dehydration-responsive element-binding protein 1A
Os03g0820400	ZFP37	C2H2型锌指转录因子ZFP37 C2H2 type zinc finger transcription factor ZFP37
Os10g0571600	OS10T0571600-01	Os10g0571600蛋白质 Os10g0571600 protein
Os01g0626400	WRKY11	WRKY转录因子11 WRKY transcription factor 11
Os03g0838800	OS03T0838800-00	锌指蛋白 Zinc finger protein
Os05g0132700	OS05T0132700-01	Os05g0132700蛋白质 Os05g0132700 protein
Os03g0287400	OsJ_10424	LOB结构域蛋白1 LOB domain protein 1
Os04g0529100	OS04T0529100-01	Os04g0529100蛋白质 Os04g0529100 protein
Os11g0154900	OS11T0154900-01	BZIP转录因子家族蛋白 BZIP transcription factor family protein
Os01g0298400	OsJ_01424	P型r2r3MYB蛋白 P-type r2r3 MYB protein

图3 相同差异表达基因分类、重要模块筛选和关键TF筛选图 A：差异表达转录因子家族分类图;B：重要模块筛选图;C：关键转录因子筛选图：蓝色上三角形为HSP70基因;黄色菱形为HSP90基因;绿色下三角为TF基因;红色长方形为关键Hsf基因

Fig. 3 Classification of the identical differentially expressed genes,important module and key TF screening A：Classification of differentially expressed TF families. B：Screening diagram of important modules. C：Screening map of key TFs：HSP70 gene is shown in the blue triangle. The yellow diamond is HSP90 gene. The green lower triangle is TF gene. The red rectangle is the key Hsf gene

图4 重要模块基因GO和KEGG富集图 A：生物功能GO富集分析;B：细胞组分GO富集分析;C：生物过程GO富集分析;D：KEGG富集分析

Fig. 4 GO and KEGG enrichment map of important module genes A：GO enrichment analysis of biological function. B：GO enrichment analysis of cell components. C：GO enrichment analysis of biological process. D：KEGG enrichment analysis

图5 盐胁迫相关基因表达验证 A：已报道盐胁迫相关基因;B：两个关键TF基因

Fig. 5 Expression verification of salt stress related genes A：Reported genes related to salt stress. B：Two key TF genes

图6 盐胁迫不同时段相关基因的表达检测 A：两个关键TF基因;B：两个HSP70基因

Fig. 6 Expression detection of related genes at different time points of salt stress A：Two key TF genes. B：Two HSP70 genes

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