基于转录组分析挖掘影响甘蓝型油菜含油量积累的候选基因

doi:10.7668/hbnxb.20195591

摘要/Abstract

摘要：

为了明晰甘蓝型油菜油脂积累的调控网络,选育高含油量油菜品种。以4个油菜自交系材料开花后25,35,45 d的种子转录组数据为样本,基于转录组和关联分析结合挖掘影响含油量的候选基因。转录组分析检测到1 530个基因在3个不同时期都存在差异表达,其中包括986个上调表达基因和544个下调表达基因。对这些差异表达基因进行GO富集分析,检测到83个油脂合成基因,79个油脂降解基因,21个油脂转运基因和80个转录因子。对差异表达转录因子进一步分析,检测到BnTT8、BnGL2、BnNAC082等基因。结合50份重测序甘蓝型油菜在2 a 3个不同区域的含油量表型,利用全基因组关联分析(GWAS)检测到BnNAC082-A03基因外显子区域的4个SNP与含油量显著关联,并检测到该基因区域存在2个单体型等位基因且BnNAC082-A03_Hap1对应材料的含油量显著高于BnNAC082-A03_Hap2对应材料。利用分析获得的转录组数据构建共表达网络,在子网络中检测到BnNAC082-A03与BnTT8-A09和BnGL2-C06直接相连,形成了影响种子油脂积累的潜在分子调控网络。

关键词: 甘蓝型油菜, 含油量, 转录组分析, 共表达网络, 全基因组关联分析

Abstract:

In order to clarify the regulatory network of oil accumulation in Brassica napus and breed oilseed rapeseed varieties with high oil content.The seed transcriptome data of four rapeseed inbred lines at 25,35,and 45 days after flowering were used to identify candidate genes affecting oil content by transcriptome analysis and correlation analysis.Consequently,a total of 1 530 genes were identified exhibiting differential expression across all three period,comprising 986 up-regulated genes and 544 down-regulated genes.GO enrichment analysis of these differentially expressed genes detected 83 lipid biosynthesis genes,79 lipid degradation genes,21 lipid transporter genes and 80 transcription factors.To further analysis of these differentially expressed transcription factors,genes including BnTT8,BnGL2,and BnNAC082 were identified.Combined with the oil content data of 50 semi-winter rapeseed in three different regions over two years,four SNPs were identified in the exons region of the BnNAC082-A03 significantly associated with oil content using genome-wide association studies.Two haplotypes were detected in the region of this gene and BnNAC082-A03_Hap1 corresponding accessions showed significantly higher oil content than that of Hap2.Additionally,it utilized these transcriptomic data to construct co-expression analysis network,and in the sub-network revealed that BnNAC082-A03 was directly connected with BnTT8-A09 and BnGL2-C06,forming a potential molecular regulatory network affecting seed lipid accumulation.

Key words: Brassica napus, Oil content, Transcriptome analysis, Co-expression networks, GWAS

中图分类号:

S565.4

李文, 姚敏, 贺丹, 邱萍, 何昕, 熊兴华, 刘忠松, 钱论文. 基于转录组分析挖掘影响甘蓝型油菜含油量积累的候选基因[J]. 华北农学报, 2025, 40(5): 47-54. doi: 10.7668/hbnxb.20195591.

LI Wen, YAO Min, HE Dan, QIU Ping, HE Xin, XIONG Xinghua, LIU Zhongsong, QIAN Lunwen. Transcriptome Analysis Reveal Candidate Genes Affecting Seed Oil Accumulation in Brassica napus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 47-54. doi: 10.7668/hbnxb.20195591.

http://www.hbnxb.net/CN/Y2025/V40/I5/47

图/表 8

表1 长沙地区4个油菜品种种子含油量

Tab.1 The oil content in seeds of four rapeseed varieties in Changsha %

品种 Varieties	含油量Oil content
品种 Varieties	15 d	20 d	25 d	30 d	35 d	40 d	45 d
XY777	2.83±0.69a	2.92±1.02a	3.36±0.48a	6.74±2.01a	31.70±1.81b	35.79±2.32b	37.36±1.67b
XY015	2.04±0.77a	2.38±0.88a	2.93±0.91a	7.74±1.33a	34.71±1.67b	39.63±1.88b	40.39±1.28b
CS136	1.48±1.32a	1.66±1.21a	2.78±1.25a	9.12±0.54a	42.54±2.12a	46.54±1.45a	49.50±1.30a
CS115	1.14±1.03a	1.32±1.03a	2.79±1.11a	8.97±0.75a	38.26±2.01a	45.01±1.01a	48.84±1.21a

图1 4个油菜品种含油量主成分分析

Fig.1 PCA of oil content between four rapeseed varieties

图2 差异表达基因数量

Fig.2 The number of differentially expressed genes

图3 差异表达基因统计及GO富集分析 A. 不同含油量品种差异表达基因Venn图;B. 3个时期差异表达Venn图。

Fig.3 Statistics of differentially expressed genes and GO enrichment analysis A. Graph of differentially expressed genes Venn in different oil content varieties; B. Venn diagram of differentially expressed in three time.

图4 差异表达基因GO富集分析 1. 有机酸生物合成过程;2. 苯丙烷分解代谢过程;3. 类黄酮生物合成过程的调控;4. 种皮发育;5. 脂肪酸运输;6. 幼苗发育;7. 对脂质的反应;8. 苯丙类生物合成过程;9. 苯丙素代谢过程;10. 脂肪酸体内平衡;11. 脂质分解代谢过程;12. 脂质氧化;13. 脂肪酸-氧化;14. 脂肪酸代谢过程;15. 原花青素的生物合成过程;16. 类黄酮代谢过程;17. 类黄酮生物合成过程;18. 蛋白质代谢过程;19. 蛋白质的运输;20. 蛋白质折叠;21. 代谢过程;22. 有机氮化合物生物合成过程;23. 有机磷代谢过程;24. 细胞组分生物发生;25. 胚胎发育结束于种子休眠;26. 脂质修饰;27. 脂肪酸分解代谢过程;28. 胚胎发育;29. 脂肪酸氧化;30. 初级代谢过程的调节;31. 核酸模板转录的调控;32. 生物合成的过程;33. 转录调控、DNA模板化;34.RNA生物合成过程的调控;35. 转录、DNA模板化;36.RNA生物合成过程;37. 基因表达;38. 光合作用;39. 有机物质的生物合成过程。

Fig.4 GO enrichment analysis of differentially expressed gene 1.Organic acid biosynthetic process;2.Phenylpropanoid catabolic process;3.Regulation of flavonoid biosynthetic process;4.Seed coat development;5.Fatty acid transport;6.Seedling development;7.Response to lipid;8.Phenylpropanoid biosynthetic process;9.Phenylpropanoid metabolic process;10.Fatty acid homeostasis;11.Lipid catabolic process;12.Lipid oxidation;13.Fatty acid beta-oxidation;14.Fatty acid metabolic process;15.Proanthocyanidin biosynthetic process;16.Flavonoid metabolic process;17.Flavonoid biosynthetic process;18.Protein metabolic process;19.Protein transport;20.Protein folding;21.Metabolic process;22.Organonitrogen compound biosynthetic process;23.Organophosphate metabolic process;24.Cellular component biogenesis;25.Embryo development ending in seed dormancy;26.Lipid modification;27.Fatty acid catabolic process;28.Embryo development;29.Fatty acid oxidation;30.Regulation of primary metabolic process;31.Regulation of nucleic acid-templated transcription;32.Biosynthetic process;33.Regulation of transcription,DNA-templated;34.Regulation of RNA biosynthetic process;35.Transcription,DNA-templated;36.RNA biosynthetic process;37.Gene expression;38.Photosynthesis;39.Organic substance biosynthetic process.

图5 转录因子表达量热图基因表达量数据转换成log₂(FPKM+1),从小到大依次利用蓝色、白色以及红色代替。

Fig.5 The heatmap displaying the expression levels of transcription factors Gene expression values are transformed into log₂(FPKM+1) replaced with blue to white to red instead from small to large.

图6 关联分析鉴定候选基因 A.围绕QTL-A03的峰值信号构建区域曼哈顿图,绿色点表示位于BnNAC082-A03基因区域的SNPs,这些SNPs与油脂含量显著关联;B.BnNAC082-A03的遗传结构变异;C.比较分析2个单体型等位基因对应材料的含油量。图中不同字母代表同一性状的不同单倍型间存在显著(P<0.05)差异。

Fig.6 Candidate genes were identified by association analysis A.Regional Manhattan plot surrounding the peak signals on QTL-A03,green dots indicate SNPs located in the BnNAC082-A03 gene regions which significantly associated with oil content; B.Genetic structure variations of BnNAC082-A03; C.Comparative analysis of the oil content of the materials corresponding to the two haplotype alleles.Different small letters indicate significant (P<0.05) between different haplotypes under the same trait.

图7 基因共表达网络 A.网络模块划分,利用不同颜色划分不同模块;B.基因网络图。根据功能标注,网络分为:转录因子(蓝色节点)、脂肪酸代谢过程(红色节点)、脂质代谢过程(橙色节点)、光合作用(紫色节点)、碳水化合物代谢过程(绿色节点)和类黄酮素代谢途径(黄色节点);C.BnNAC082-A03表达量(FPKM)与油脂含量之间的相关性;D.BnTT8-A09表达量(FPKM)与油脂含量之间的相关性;E.BnGL2-C06表达量(FPKM)与油脂含量之间的相关性。

Fig.7 Gene co-expression networks A.Network module partitioning,using different colors to delineate different modules; B.Gene network diagram.According to functional labeling,networks are divided into:transcription factor (blue nodes),fatty acid biosynthesis (red nodes),lipid biosynthesis (orange nodes),photosynthesis (purple nodes),carbohydrate metabolism (green nodes) and flavonoid biosynthetic (yellow nodes); C.Correlation between the expression (FPKM) of BnNAC082-A03 with oil content; D.Correlation between the expression (FPKM) of BnTT8-A09 with oil content; E.Correlation between the expression (FPKM) of BnGL2-C06 with oil content.

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