‘红地球’葡萄花芽分化过程中的转录组分析

doi:10.3864/j.issn.0578-1752.2022.20.013

摘要/Abstract

摘要：

【目的】葡萄是我国重要的果树树种,花芽分化直接影响葡萄的质量和数量。对‘红地球’葡萄花芽分化过程中的花芽进行比较分析,探索‘红地球’葡萄花芽分化机制,挖掘关键基因,为了解‘红地球’葡萄花芽分化提供理论基础。【方法】对‘红地球’葡萄花芽分化过程中4个发育阶段：S1（未分化期）、S2（花原始体发育期）、S3（花序主轴发育期）和S4（花序二级轴发育期）的芽进行形态学观察和植物激素测定,并进行转录组测序分析及验证。【结果】 ‘红地球’葡萄花芽分化过程中共发现13 729个差异基因,其中S1-S2、S2-S3、S3-S4和S1-S4分别有4 158、2 050、3 425和7 652个差异基因。在S1-S4差异基因的富集调控网络中发现差异基因在激素介导的信号通路、脱落酸代谢过程、对酸性化学物质的反应和植物细胞壁组织或生物发生等通路富集。在激素介导的信号通路中发现大量与生长素、赤霉素和脱落酸等相关基因,测定表明,生长素在S2时期含量最高,而在S3和S4时期含量最低;赤霉素含量在花芽分化过程中不断降低,在S4时期为S1时期的80%;脱落酸含量在S1和S4时期较高,而在S2时期最低。此外,S1-S4差异基因来自转录因子家族（MYB、ERF、bHLH和MADS-box等）,表明这些转录因子家族基因参与了‘红地球’葡萄花芽分化。对差异表达的13个MADS-box家族基因进一步分析表明,MADS8、AGL65、AGL15、AGL12和MADS2在花芽分化进程中表达上调,而AGL30、LeMADS、FBP24、AGL14和MADS3表达下调。对这些MADS-box基因进行qRT-PCR验证,基因表达趋势与转录组数据一致且相关系数较高,表明数据分析结果可靠。【结论】 ‘红地球’葡萄花芽分化是一个复杂的生物过程,其中,植物激素介导的信号通路以及MADS-box家族基因在花芽分化中发挥重要作用。研究结果提供了一个关于转录因子、基因和激素的信息,有助于揭开这一复杂的发育过程,并为‘红地球’葡萄花芽分化综合模型的建立提供理论基础。

关键词: ‘红地球’葡萄, 花芽分化, 转录组测序, 植物激素, MADs-box

Abstract:

【Objective】 Grape is an important fruit tree species of China, and the flower bud differentiation directly affects the quality and quantity of this grape. In this study, Red Globe grape developmental stages during flower bud differentiation under natural growth conditions were compared and analyzed, the mechanism of flower bud differentiation of this grape variant was evaluated, and the essential genes were mined, so as to provide a theoretical basis for understanding the flower bud differentiation of this grape species. 【Method】During flower bud differentiation of the Red Globe grape, the morphological observation, phytohormone determination, and transcriptome sequencing analysis were performed on the buds of four developmental stages, including S1 (undifferentiated), S2 (flower anlagen development), S3 (formation of the main cob of inflorescence), and S4 (formation of the second cob of inflorescence). 【Result】A total of 13 729 differentially expressed genes were determined during the flower bud differentiation process of the ‘Red Globe’ grape, which included 4 158, 2 050, 3 425, and 7 652 genes in S1-S2, S2-S3, S3-S4, and S1-S4, respectively. In the enrichment-regulation network of the S1-S4 differential genes, those differential genes were found to be enriched in the hormone-mediated signaling pathways, abscisic acid metabolism, acid chemical reactions, plant cell wall tissues, or biogenesis. Several genes related to auxin, gibberellin, and abscisic acid were detected in the hormone-mediated signaling pathway. The results revealed that the content of auxin was the highest in S2 and the lowest in S3 and S4. The gibberellin content decreased continuously during flower bud differentiation, 80% of that at S4 in S1; the abscisic acid content was higher in S1 and S4 and the lowest in S2. In addition, the S1-S4 differential genes belonged to the transcription factor families (MYB, ERF, bHLH, and MADS-box), indicating that these family genes were involved in the flower bud differentiation of the Red Globe grape. The further analysis of the 13 differentially expressed MADS-box genes revealed upregulated expressions of MADS8, AGL65, AGL15, AGL12, and MADS2 during flower bud differentiation. In contrast, the expressions of AGL30, LeMADS, FBP24, AGL14, and MADS3 were downregulated. These MADS-box genes were verified via quantitative reverse transcription polymerase chain reaction, and the expression trend was found to be consistent with the corresponding transcriptome. 【Conclusion】The flower bud differentiation of the Red Globe grape was a complex biological process. The plant hormone-mediated signaling pathways and the MADS-box family genes played essential roles in flower bud differentiation. These results provided information about transcription factors, genes, and hormones to help understand this complex developmental process and provided a theoretical basis for establishing a comprehensive model for flower bud differentiation in the Red Globe grape.

Key words: Red Globe grapes, flower bud differentiation, transcriptome sequencing, plant hormone, MADs-box

刘鑫,张亚红,袁苗,党仕卓,周娟. ‘红地球’葡萄花芽分化过程中的转录组分析[J]. 中国农业科学, 2022, 55(20): 4020-4035.

LIU Xin,ZHANG YaHong,YUAN Miao,DANG ShiZhuo,ZHOU Juan. Transcriptome Analysis During Flower Bud Differentiation of Red Globe Grape[J]. Scientia Agricultura Sinica, 2022, 55(20): 4020-4035.

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基因名称 Gene name	基因号 Gene ID	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
MADS8	VIT_14s0068g01800	AGCGACGAAGCGGAATGATGAAG	GGCGGAGAAGATGATCAGAGCAAG
AGL65	VIT_17s0000g06340	GGGTAGGGTGAGGAGGAATGGATC	ATGGACGGTGGGCTTTGTGAATG
AGL30	VIT_04s0008g01980	TTCTCAGCGGAAGCAAGGCATTC	GGCGAGAACATGAGGAGCAAGAC
AGL21	VIT_00s0211g00180	AGAATCTAGAGCGAGCCGTGAGG	GCCTTCTTCAACAGTCCCTTCCTTC
AGL21	VIT_18s0001g07900	CAACGGGATGGGTTTGGGATGAG	ACGAGACAAGGGAGACGAGTATGG
AGL15	VIT_13s0158g00100	CCACTCATCCACCACTTCCATTCC	GTGCCTTGATGCTGACCTACCTAC
LeMADS	VIT_03s0017g00360	TCTCTGTGATGCTGATGTTGCTCTC	GGTCCCCGGAATTTCACTGAGTATG
FBP24	VIT_02s0025g02350	CAGGCAACCACAACCAAATCAAACC	ACTGAGGAGGGATGGGCACAAG
AGL14	VIT_15s0048g01240	CAACTGGGTTTCTGGAGATGGTGAG	AAAGGTGACTTGCCGACTTGTGG
AGL12	VIT_18s0041g02140	GCGTATTGAGAACCCAGTACACAGG	TCAGCATCACACAACACAGAGAGC
MADS3	VIT_16s0022g02330	AGAGAACAAGATCAACCGCCAAGTC	TCAGCATCGCAGAGCACAGAAAG
MADS2	VIT_14s0083g01050	TTCCACACTGCCTATGTTGAACCTG	GGACGGTGCGATTAGAGCCAAC
MADS2	VIT_17s0000g05000	TTCCACACTGCCTATGTTGAACCTG	GGACGGTGCGATTAGAGCCAAC
Actin1	XP_008654957.1	TCCTTGCCTTGCGTCATCTAT	CACCAATCACTCTCCTGCTACAA

基因名称 Gene name	基因号 Gene ID	转录因子 TF id	蛋白质长度 Protein length (aa)	家族 Family
MADS8	VIT_14s0068g01800	GSVIVT01001437001	249	MIKC
AGL65	VIT_17s0000g06340	GSVIVT01003864001	225	MIKC
AGL30	VIT_04s0008g01980	GSVIVT01008139001	247	MIKC
AGL21	VIT_00s0211g00180	GSVIVT01009219001	241	MIKC
AGL21	VIT_18s0001g07900	GSVIVT01015641001	218	MIKC
AGL15	VIT_13s0158g00100	GSVIVT01018450001	250	MIKC
LeMADS	VIT_03s0017g00360	GSVIVT01019630001	597	MIKC
FBP24	VIT_02s0025g02350	GSVIVT01025916001	199	MIKC
AGL14	VIT_15s0048g01240	GSVIVT01027579001	219	MIKC
AGL12	VIT_18s0041g02140	GSVIVT01036551001	244	MIKC
MADS3	VIT_16s0022g02330	GSVIVT01007989001	87	Mδ
MADS2	VIT_14s0083g01050	GSVIVT01033067001	376	Mα
MADS2	VIT_17s0000g05000	GSVIVT01035477001	184	Mδ

样本 Sample	纯净数据 Clean reads	Q30含量 Q30 (%)	GC含量 GC content (%)	总读取数 Total reads	映射总数 Total mapped
S1-1	56701744	93.50	46.08	56701744	51584717 (90.98%)
S1-2	57000078	93.57	46.30	57000078	52047737 (91.31%)
S1-3	53106034	93.75	46.23	53106034	48593610 (91.5%)
S2-1	57074664	93.28	45.85	57074664	52283600 (91.61%)
S2-2	48571060	93.29	45.78	48571060	44806530 (92.25%)
S2-3	52541122	93.72	45.81	52541122	48822966 (92.92%)
S3-1	44482618	93.74	45.55	44482618	41222350 (92.67%)
S3-2	44016334	93.83	45.42	44016334	40718241 (92.51%)
S3-3	43775084	93.97	45.94	43775084	40797167 (93.2%)
S4-1	43004638	93.77	46.42	43004638	33306442 (77.45%)
S4-2	48834670	93.72	45.92	48834670	45022453 (92.19%)
S4-3	51647760	93.52	46.39	51647760	46614037 (90.25%)