乌塌菜雄性不育关键基因的鉴定及功能分析

doi:10.16420/j.issn.0513-353x.2024-0923

摘要/Abstract

摘要：

以乌塌菜（Brassica campestris L.）可育系和细胞质雄性不育系植株为试材，利用转录组学与蛋白质组学分析，共鉴定出6 657个差异表达基因（DEG）和1 443个差异表达蛋白（DEP），主要富集在以下过程/通路中：花粉发育（GO：0009555）和花粉壁组装（GO：0010208）等生物过程，以及苯丙素生物合成（ko00940）、角质、木栓质和蜡质生物合成（ko00073）等代谢途径。通过联合分析，发现转录组与蛋白组之间呈现正相关，共有387个mRNA与蛋白关联在转录水平和蛋白丰度上均表现出显著差异。对富集在花粉发育与花粉壁组装生物过程中的蛋白进行蛋白—蛋白互作（PPI）网络分析，发现BraA05g034940.3C（FAR2）等蛋白与其他蛋白关联较强。BcFAR2在花蕾组织中，尤其是花药发育的四分体与单核期表达量较高，并且过表达BcFAR2可恢复拟南芥far2突变体的花粉育性。

关键词: 乌塌菜, 转录组, 蛋白质组, 雄性不育, BcFAR2, 功能分析

Abstract:

A total of 6 657 differentially expressed genes（DEG）and 1 443 differentially expressed proteins（DEP）were identified between fertile and sterile Wucai（Brassica campestris L.）plants，based on transcriptomic and proteomic analyses. The DEG and DEP were primarily enriched in biological processes related to pollen development（GO：0009555）and pollen wall assembly（GO：0010208），as well as metabolic pathways including phenylpropanoid biosynthesis（ko00940），and cutin，suberin and wax biosynthesis（ko00073）. An integrated analysis identified 387 mRNA-protein association pairs showing a positive correlation between transcriptome and proteome data. Protein-protein interaction（PPI）network analysis of proteins involved in the pollen development and pollen wall assembly revealed that several proteins，including BraA05g034940.3C（FAR2），were highly interconnected with other proteins. Expression pattern analysis indicated that BcFAR2 gene was most highly expressed in floral bud tissue，particularly during the tetrad and mononucleate stages of anther development. Moreover，overexpression of BcFAR2 restored pollen fertility in Arabidopsis far2 mutants.

Key words: Wucai, transcriptome, proteome, male sterility, BcFAR2, functional analysis

冯克政, 王康, 汪永康, 赵龙龙, 廖娜, 唐小燕, 王文杰, 汪承刚, 袁凌云, 侯金锋, 吴建强, 陈国户. 乌塌菜雄性不育关键基因的鉴定及功能分析[J]. 园艺学报, 2025, 52(11): 2876-2888.

FENG Kezheng, WANG Kang, WANG Yongkang, ZHAO Longlong, LIAO Na, TANG Xiaoyan, WANG Wenjie, WANG Chenggang, YUAN Lingyun, HOU Jinfeng, WU Jianqiang, CHEN Guohu. Identification and Functional Analysis of Key Gene for Male Sterility in Wucai[J]. Acta Horticulturae Sinica, 2025, 52(11): 2876-2888.

图/表 11

表1 引物序列

Table 1 Primer sequences

用途 Application	基因ID/引物名称 Gene ID/Primer name	引物（5′-3′） Primer sequence
克隆 Gene cloning	BcFAR2	F：ATGGAAGCTCTCTTCTTGAC	R：TTAAGCTCTTCCCTTCAAG
荧光定量PCR qRT-PCR	BraA01g038270.3C	F：AGGACTGGACCCTTTCATGG	R：CTAAGGTGCGCTTCTGTGTC
	BraA02g023150.3C	F：TGTGTCACGATGGACGATCA	R：GAGCTCGTTCTGGTCAAACC
	BraA03g045620.3C	F：CAAATGGCTCAGACCACACC	R：TTCGTGTCACAATTGGTGCT
	BraA04g008040.3C	F：TTTGGCTCCGGGAAGAGAAT	R：TCGAGCCAGTCCATTGAAGT
	BraA04g026900.3C	F：CAGGAAAGGCAACAGTGCTT	R：TGTAGCGGGTCTTCACAGTT
	BraA05g034940.3C	F：TGCTAAGCATGGAGTGGTGA	R：AAGAAGCTCTGCCAAGTCCT
	BraA09g012710.3C	F：GAGAATGTGGCGTTCGTTGA	R：ATCCCATACTCGGCGACATT
	BraA09g066080.3C	F：TATGGGACGACACTGACGAG	R：GCACCATCGTCACACCTAAC
	Actin BraA05g007930.3C	F：TGGGTTTGCTGGTGACGAT	R：TGCCTAGGACGACCAACAATACT
载体构建 Vector construction	pCAMBIA1305-FAR2	F：gacgatgacgataagggatccATGGAAGCTCTCTTCTTGACTTCTTC R：tgcctgcaggtcgactctagaTTAAGCTCTTCCCTTCAAGACATG
突变体鉴定 Mutant identification	far2	LP：GTTTTATGTTACGGGAAGGGG
		RP：GCATTACTATGAGAAGAGACTTTGCC
		LB：ATTTTGCCGATTTCGGAAC

图1 乌塌菜细胞质雄性不育系12-14A与可育系12-14B花特征比较

Fig. 1 Comparison of Wucai flower characteristics between sterile line 12-14A and fertile line 12-14B

图2 乌塌菜不育与可育植株四分体时期花蕾转录组分析 A：DEG表达聚类热图（MS：不育植株；MF：可育植株）；B：差异表达基因GO富集分析；C：差异表达基因KEGG富集分析

Fig. 2 Transcriptome analysis of Wucai flower buds in sterile and fertile plants at the tetrad stage A：Heatmap of DEG（MS：male sterile；MF：male fertile）；B：GO enrichment analysis of DEG；C：KEGG enrichment analysis of DEG

图3 乌塌菜不育与可育植株四分体时期花蕾蛋白质组分析 A：DEP表达聚类热图（MS：不育植株；MF：可育植株）；B：差异表达蛋白GO富集分析；C：差异表达蛋白KEGG富集分析

Fig. 3 Proteome analysis of Wucai flower buds in sterile and fertile plants at the tetrad stage A：Heatmap of DEP（MS：male sterile；MF：male fertile）；B：GO enrichment analysis of DEP；C：KEGG enrichment analysis of DEP

图4 乌塌菜转录组与蛋白质组联合分析 A：差异表达基因与差异表达蛋白Venn图；B：四象限图，共鉴定出2 266个可定量到的蛋白-mRNA对，NDEP表示蛋白水平不差异，NDEG表示转录水平不差异；C：GO富集分析mRNA与蛋白关联基因对；D：KEGG富集分析mRNA与蛋白关联基因对；E：花粉发育发育通路相关基因表达分析；F：花粉发育发育通路相关基因PPI网络图

Fig. 4 Integrated analysis of transcriptome and proteome A：Venn diagram of overlapping DEG and DEP；B：Four-quadrant plot of DEG and DEP expression correlation，a total of 2 266 quantifiable mRNA-protein pairs were identified，NDEP indicates no difference in protein level，NDEG indicates no difference in transcription level；C：GO enrichment analysis of mRNA-protein association pairs；D：KEGG enrichment analysis of mRNA-protein association pairs；E：Heatmap of genes involved in pollen development pathway；F：Protein-protein interaction（PPI）network of genes associated with pollen development pathway

表2 mRNA与蛋白关联基因GO与KEGG富集分析结果（前10）

Table 2 GO and KEGG enrichment results for mRNA-protein association genes（top 10）

GO术语/KEGG通路 GO term/KEGG pathway	注释 Description	分类 Class	数量 Number	Q值 Q-value
GO：0010208	花粉壁组装Pollen wall assembly	生物过程 Biological process	27	2.11E-26
GO：0010927	参与形态发生的细胞成分组装 Cellular component assembly involved in morphogenesis		27	2.11E-26
GO：0085029	细胞外基质组装Extracellular matrix assembly		27	2.11E-26
GO：0030198	细胞外基质组织Extracellular matrix organization		27	4.19E-26
GO：0043062	细胞外结构组织Extracellular structure organization		27	4.19E-26
GO：0009555	花粉发育Pollen development		27	6.28E-24
GO：0048646	参与形态发生的解剖结构形成 Anatomical structure formation involved in morphogenesis		27	5.11E-19
GO：0048229	配子体发育Gametophyte development		30	7.80E-14
GO：0045229	外部封装结构组织External encapsulating structure organization		40	1.02E-11
GO：0032989	细胞成分形态发生Cellular component morphogenesis		34	7.94E-10
ko00940	苯丙素生物合成Phenylpropanoid biosynthesis	代谢 Metabolism	12	4.52E-2
ko00073	角质、木栓质和蜡生物合成Cutin，suberine and wax biosynthesis		6	4.52E-2
ko00196	光合作用—天线蛋白Photosynthesis-antenna proteins		6	4.52E-2
ko00010	糖酵解/糖异生Glycolysis/gluconeogenesis		15	6.68E-2
ko00053	抗坏血酸和醛糖代谢Ascorbate and aldarate metabolism		6	3.70E-2
ko00310	赖氨酸降解Lysine degradation		5	3.70E-2
ko00280	缬氨酸、亮氨酸和异亮氨酸降解 Valine，leucine and isoleucine degradation		7	5.57E-2
ko00620	丙酮酸代谢Pyruvate metabolism		10	5.67E-2
ko00052	半乳糖代谢Galactose metabolism		6	5.67E-2
ko04626	植物—病原体相互作用Plant-pathogen interaction	生物系统 Organismal systems	9	5.67E-2

表2 mRNA与蛋白关联基因GO与KEGG富集分析结果（前10）

Table 2 GO and KEGG enrichment results for mRNA-protein association genes（top 10）

GO术语/KEGG通路 GO term/KEGG pathway	注释 Description	分类 Class	数量 Number	Q值 Q-value
GO：0010208	花粉壁组装Pollen wall assembly	生物过程 Biological process	27	2.11E-26
GO：0010927	参与形态发生的细胞成分组装 Cellular component assembly involved in morphogenesis		27	2.11E-26
GO：0085029	细胞外基质组装Extracellular matrix assembly		27	2.11E-26
GO：0030198	细胞外基质组织Extracellular matrix organization		27	4.19E-26
GO：0043062	细胞外结构组织Extracellular structure organization		27	4.19E-26
GO：0009555	花粉发育Pollen development		27	6.28E-24
GO：0048646	参与形态发生的解剖结构形成 Anatomical structure formation involved in morphogenesis		27	5.11E-19
GO：0048229	配子体发育Gametophyte development		30	7.80E-14
GO：0045229	外部封装结构组织External encapsulating structure organization		40	1.02E-11
GO：0032989	细胞成分形态发生Cellular component morphogenesis		34	7.94E-10
ko00940	苯丙素生物合成Phenylpropanoid biosynthesis	代谢 Metabolism	12	4.52E-2
ko00073	角质、木栓质和蜡生物合成Cutin，suberine and wax biosynthesis		6	4.52E-2
ko00196	光合作用—天线蛋白Photosynthesis-antenna proteins		6	4.52E-2
ko00010	糖酵解/糖异生Glycolysis/gluconeogenesis		15	6.68E-2
ko00053	抗坏血酸和醛糖代谢Ascorbate and aldarate metabolism		6	3.70E-2
ko00310	赖氨酸降解Lysine degradation		5	3.70E-2
ko00280	缬氨酸、亮氨酸和异亮氨酸降解 Valine，leucine and isoleucine degradation		7	5.57E-2
ko00620	丙酮酸代谢Pyruvate metabolism		10	5.67E-2
ko00052	半乳糖代谢Galactose metabolism		6	5.67E-2
ko04626	植物—病原体相互作用Plant-pathogen interaction	生物系统 Organismal systems	9	5.67E-2

图5 乌塌菜花粉发育相关基因表达分析热图表示基于蛋白质组数据的蛋白丰度；数值为log2（count），并已均一化；上层颜色条表示蛋白丰度水平值

Fig. 5 Expression analysis of Wucai genes associated with pollen development Heatmaps show protein abundance in sterile and fertile lines based on transcriptome analysis；values are log2（count）and normalized，with color bars on the top representing protein abundance values

图6 乌塌菜BcFAR2序列比对蓝色框为FAR-N（CCD：cd05236），绿色框为FAR-C（CCD：cd09071），红色下划线表示NAD结合位点（PF07933；雄心不育蛋白）；AtFAR2：拟南芥（AT3G11980）；BcFAR2：乌塌菜；BniFAR2：黑芥（BniB01g052980.2N）；BoFAR2：甘蓝（BolC05g054690.2J）；BrFAR2：大白菜（BraA05g034940.3C）

Fig. 6 Sequence alignment of the BcFAR2 in Wucai The blue box indicates the FAR-N domain（CCD：cd05236），the green box indicates the FAR-C domain（CCD：cd09071），and the red line indicates the NAD binding site（PF07933；Male sterility protein）；AtFAR2，A. thaliana（AT3G11980）；BcFAR2：Brassica campestris；BniFAR2：B. nigra（BniB01g052980.2N）；BoFAR2：B. oleracea（BolC05g054690.2J）；BrFAR2：B. rapa（BraA05g034940.3C）

图7 乌塌菜BcFAR2亚细胞定位分析 A：亚细胞定位载体；B：亚细胞定位

Fig. 7 Subcellular localization analysis of BcFAR2 A：Vector construction for subcellular localization. B：Subcellular localization

图8 乌塌菜BcFAR2基因表达分析 A：BcFAR2在乌塌菜各组织中的相对表达量；B：BcFAR2在乌塌菜可育植株不同花药发育时期中的表达量；C：乌塌菜不育植株中BcFAR2在不同花药发育时期相对可育植株的表达量。柱上不同小写字母表示Duncan’s多重检验在P < 0.05水平上差异显著

Fig. 8 Expression analysis of Wucai BcFAR2 gene A：Expression of BcFAR2 in various tissues of Wucai. B：Expression of BcFAR2 in different stages of anther development in fertile Wucai. C：Relative expression of BcFAR2 at various anther development stages in sterile versus fertile lines. Different lowercase letters indicate significant differences as determined by Duncan’s new multiple range test（p < 0.05）

图9 乌塌菜BcFAR2基因功能分析 A：BcFAR2遗传转化拟南芥far2突变体表型；B：拟南芥野生型Col-0、far2突变体及far2/35S::BcFAR2植株花粉活力分析

Fig. 9 Functional analysis of Wucai BcFAR2 gene A：Phenotypic of the Arabidopsis far2 mutant transformed with the BcFAR2 gene. B：Pollen viability analysis of Arabidopsis thaliana Col-0，far2 mutant and far2/35S::BcFAR2 plants

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