Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (11): 2876-2888.doi: 10.16420/j.issn.0513-353x.2024-0923
• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles
FENG Kezheng1,2, WANG Kang1,2, WANG Yongkang1,2, ZHAO Longlong1,2, LIAO Na1,2, TANG Xiaoyan1,2, WANG Wenjie1,2, WANG Chenggang1,2, YUAN Lingyun1,2, HOU Jinfeng1,2, WU Jianqiang1,2, and CHEN Guohu1,2,*(
)
Received:2025-05-21
Revised:2025-09-24
Online:2025-11-26
Published:2025-11-26
Contact:
and CHEN Guohu
FENG Kezheng, WANG Kang, WANG Yongkang, ZHAO Longlong, LIAO Na, TANG Xiaoyan, WANG Wenjie, WANG Chenggang, YUAN Lingyun, HOU Jinfeng, WU Jianqiang, and CHEN Guohu. Identification and Functional Analysis of Key Gene for Male Sterility in Wucai[J]. Acta Horticulturae Sinica, 2025, 52(11): 2876-2888.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2024-0923
| 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 | |||
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 | |||
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
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
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
| 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 |
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 |
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
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)
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)
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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