刺梨SOD基因家族鉴定与表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1138

摘要/Abstract

摘要：

【目的】超氧化物歧化酶（SOD）是植物体内氧自由基的天然清除剂，对于保护植物免受环境胁迫起着关键作用，了解其在刺梨响应干旱胁迫中的作用，为深入研究刺梨SOD基因家族的功能提供基础。【方法】通过生物信息学对刺梨SOD基因家族进行系统鉴定和分析，对其理化性质、染色体位置、基因结构、亚家族进化、顺式作用元件和WGCNA进行全面分析，并利用RT-qPCR分析刺梨SOD基因家族在干旱胁迫下的表达模式。【结果】刺梨SOD基因家族有9个成员，包括4个Cu/ZnSOD基因、3个MnSOD基因和2个FeSOD基因，分布在5条染色体上。基因结构显示，同一亚家族成员的基序组成较为相似，但内含子/外显子排列和数量差异较大。亚家族进化分析发现，MnSOD亚家族较原始，其在所有蛋白质位置都表现出高度保守性，其次是FeSOD和Cu/ZnSOD，Cu/ZnSOD亚家族各序列之间差异较大，又可以分为3个亚类。顺式作用元件分析显示，该家族基因参与多种植物激素、生长发育以及胁迫响应，特别是RrCSD2启动子区含有类黄酮生物合成元件（MBSI）。进一步通过转录组和WGCNA分析发现，RrCSD2所在模块的基因显著富集到黄酮和黄酮醇生物合成、类黄酮生物合成和花生四烯酸代谢等途径，表明RrCSD2可能参与类黄酮代谢过程。RT-qPCR结果显示，在干旱胁迫下，除RrMSD1和RrMSD2的表达水平显著下降外，其余7个基因的表达水平总体呈现上升趋势，特别是RrCSD2和RrCSD3的表达水平显著上调。【结论】刺梨SOD基因在干旱胁迫中起到重要作用。

关键词: 刺梨, SOD基因, 亚家族进化, WGCNA分析, 干旱胁迫

Abstract:

【Objective】Superoxide dismutase（SOD）is a natural scavenger of oxygen free radicals in plants, which plays a crucial role in protecting plants from environmental stress. The aim of this study is to investigate the role of SOD in response to drought stress in Rosa roxburghii and to provide a basis for further research on the function of the SOD gene family in Rosa roxburghii. 【Method】The SOD gene family of R. roxburghii was systematically identified and analyzed by bioinformatics method. The physicochemical properties, chromosome location, gene structure, subfamily evolution, cis-acting elements and WGCNA were comprehensively analyzed. Additionally, the expression pattern of the SOD gene family of R. roxburghii under drought stress was analyzed by RT-qPCR. 【Result】There was a total of nine members of SOD gene family in R. roxburghii, including four Cu/ZnSOD genes, three MnSOD genes, and two FeSOD genes. These genes were scattered across five chromosomes. The analysis of gene structure indicated that members within the same subfamily had similar motifs, but there were notable variations in the number and arrangement of introns/exons. Subfamily evolution analysis demonstrated that the MnSOD subfamily was the most primitive, with high conservation across all protein positions, followed by the FeSOD and Cu/ZnSOD subfamilies. The Cu/ZnSOD subfamily could be further divided into three subclasses due to significant sequence differences. Cis-element analysis revealed the involvement of the SOD gene family in various plant hormones, growth, and stress responses. Notably, the promoter region of RrCSD2 contained flavonoid biosynthetic elements（MBSI）. Transcriptome and WGCNA analysis further revealed that the genes of RrCSD2 module were significantly enriched in flavonoid and flavonol biosynthesis, as well as flavonoid biosynthesis and arachidonic acid metabolism, suggesting that RrCSD2 may be involved in flavonoid metabolism. RT-qPCR results showed that the expressions of RrMSD1 and RrMSD2 significantly decreased under drought stress, while the expressions of the other seven genes increased overall. Specifically, the expressions of RrCSD2 and RrCSD3 were significantly up-regulated. 【Conclusion】The SOD genes of R. roxburghii play a crucial role in drought stress response.

Key words: Rosa roxburghii, SOD gene, subfamily evolution, WGCNA analysis, drought stress

文洁, 杜元欣, 吴安波, 杨广容, 鲁敏, 安华明, 南红. 刺梨SOD基因家族鉴定与表达模式分析[J]. 生物技术通报, 2024, 40(5): 153-166.

WEN Jie, DU Yuan-xin, WU An-bo, YANG Guang-rong, LU Min, AN Hua-ming, NAN Hong. Identification and Expression Pattern Analysis of Rosa roxburghii SOD Gene Family[J]. Biotechnology Bulletin, 2024, 40(5): 153-166.

图/表 13

表1 RT-qPCR引物

Table 1 Primers used for RT-qPCR

基因名称Gene name	基因ID Gene ID	上游引物Upstream primer（5'-3'）	下游引物Downstream primer（5'-3'）
RrCSD1	evm.model.Contig149.101	GATGGCCCAACTACTGTGACT	ACCGTTTGTTGTGTCACCAAG
RrCSD2	evm.model.Contig172.106	CTGTATTGCCAGAGCTACTGAC	TGCACCCAGAATCCTAACAAC
RrCSD3	evm.model.Contig255.128	CCGGTGATTTGGGTAACGTCACT	GTTCATGTCCACCTGCCAGTCGTC
RrCSD4	evm.model.Contig428.424	ACCCAAGAAGATGACGGTCCT	TCGTGTCACCAAACTCATGCAAG
RrMSD1	evm.model.Contig308.148	ATCATGGACCTCCATCACTT	GTGGAGGAGTCAACTGTATC
RrMSD2	evm.model.Contig308.149	GATACGGAAGGAGGAGGTGA	GAACCTTGCAAAGAAGCACC
RrMSD3	evm.model.Contig317.61	GCACCACCAGGCTTACATCACC	CGCTCTGCAACTTAACGACGG
RrFSD1	evm.model.Contig217.78	TGAAGCCTCCTCCGTATCCA	TATCCACATAACCCCTATGGTG
RrFSD2	evm.model.Contig285.37	AACCATCTTGTGTCTTGGAA	AACAGGGATATTAGGTTCGC
UBQ	-	ATGCAGATTTTGTGAAGAC	ACCACCACGRAGACGGAG

表2 RrSOD基因理化性质

Table 2 Physical and chemical properties of RrSOD genes

基因名称 Gene name	基因ID Gene ID	氨基酸 Amino acid/aa	分子量 Molecular weight/Da	等电点 pI	亚细胞定位 Location
RrCSD1	evm.model.Contig149.101	161	16 424.23	5.81	线粒体Mitochondria
RrCSD2	evm.model.Contig172.106	252	26 826.36	4.82	线粒体Mitochondria
RrCSD3	evm.model.Contig255.128	126	12 912.35	5.68	过氧化物酶体Peroxysome
RrCSD4	evm.model.Contig428.424	224	23 278.95	5.79	叶绿体Chloroplast
RrMSD1	evm.model.Contig308.148	196	21 612.59	7.73	线粒体Mitochondria
RrMSD2	evm.model.Contig308.149	235	26 494.16	6.16	线粒体Mitochondria
RrMSD3	evm.model.Contig317.61	180	19 243.86	7.07	线粒体Mitochondria
RrFSD1	evm.model.Contig217.78	268	30 684.68	5.43	线粒体Mitochondria
RrFSD2	evm.model.Contig285.37	270	31 123.38	6.33	线粒体Mitochondria

图1 RrSOD基因在染色体上的分布

Fig. 1 Locations of RrSOD genes on chromosomes

表3 野草莓与月季SOD基因家族成员

Table 3 SOD gene family members of F. vesca and R. chinensis

序号No.	基因ID Gene ID	物种Species	基因名称Gene name	分类Classifying
1	FvH4_6g04231.t2	野草莓F. vesca	FvSOD1	Cu/ZnSOD
2	FvH4_3g30620.t1		FvSOD2	Cu/ZnSOD
3	FvH4_1g28280.t1		FvSOD3	Cu/ZnSOD
4	FvH4_5g01090.t1		FvSOD4	Cu/ZnSOD
5	FvH4_7g25730.t1		FvSOD5	MnSOD
6	FvH4_4g37110.t1		FvSOD6	FeSOD
7	FvH4_7g26501.t1		FvSOD7	FeSOD
8	RchiOBHm_Chr3g0452901	月季R. chinensis	RcSOD1	Cu/ZnSOD
9	RchiOBHm_Chr5g0056701		RcSOD2	Cu/ZnSOD
10	RchiOBHm_Chr3g0496871		RcSOD3	Cu/ZnSOD
11	RchiOBHm_Chr2g0126391		RcSOD4	Cu/ZnSOD
12	RchiOBHm_Chr1g0371971		RcSOD5	MnSOD
13	RchiOBHm_Chr4g0446901		RcSOD6	FeSOD
14	RchiOBHm_Chr5g0072741		RcSOD7	MnSOD
15	RchiOBHm_Chr1g0373101		RcSOD8	FeSOD
16	RchiOBHm_Chr1g0318061		RcSOD9	FeSOD

图2 SOD基因家族系统进化分析 A：刺梨、月季和野草莓SOD基因家族共线性分析；B：4个物种的SOD基因家族系统进化分析。At：拟南芥，Rr：刺梨，Fv：野草莓，Rc：月季。利用MEGA X软件构建系统进化树（邻接法），步数设置为1 000

Fig. 2 Phylogenetic analysis of SOD gene family A: Colinear analysis of SOD gene family in R. roxburghii, R. chinensis, and F. vesca. B: Phylogenetic analysis of SOD gene family in four species. At: A. thaliana, Rr: R. roxburghii, Fv: F. vesca, Rc: R. chinensis. The phylogenetic tree was construct using MEGA X with neighbor-joining method, and bootstrap was set to 1 000

图3 RrSOD基因内含子-外显子结构

Fig. 3 Exon-intron structures of RrSOD genes

图4 RrSOD基因保守基序

Fig. 4 Conservative motifs of RrSOD genes

图5 蔷薇科SOD基因亚家族进化分析 A：SOD亚家族保守位点分布热图；B：SOD亚家族特异位点分布图；C：SOD亚家族20个最保守特异位点Weblog图；D：SOD亚家族UMAP聚类图；E：SOD亚家族同源聚类图；F：蔷薇科基因谱系关系

Fig. 5 Analysis of SOD gene subfamily evolution in Rosaceae A: Heatmap showing the conservation of each site in three SOD subfamilies. B: Distribution of the specific sites in three SOD subfamilies. C: Weblog of the 20 most conserved specific site of each SOD subfamily. D: UMAP clustering diagram of SOD subfamilies. E: SOD subfamily homology order. F: Gene lineage relationships of Rosaceae

图6 RrSOD基因顺式作用元件预测

Fig. 6 Prediction of cis-acting elements of RrSOD genes

图7 RrSOD基因上游1 500 bp顺式作用元件分布

Fig. 7 Distribution of cis-acting elements in the 1 500 bp upstream region of RrSOD genes

图8 刺梨花、叶、茎和果实不同发育阶段RrSOD基因表达

Fig. 8 Expressions of RrSOD genes in the flowers, leaves, stems and fruits at different developmental stages of R. roxburghii

图9 RrSOD基因WGCNA分析 A：共表达模块与层次聚类树；B：KEGG富集分析

Fig. 9 WGCNA analysis of RrSOD genes A: Co-expression module and hierarchical clustering tree. B: KEGG enrichment analysis

图10 干旱胁迫下RrSOD基因的相对表达分析不同小写字母表示在0.05水平差异显著

Fig. 10 Relative expression analysis of RrSOD genes under drought stress Different lowercase letters indicate significant difference at 0.05 level

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