葡萄不同性别花器官发育机制初探

doi:10.16420/j.issn.0513-353x.2023-0010

摘要/Abstract

摘要：

以‘北醇'葡萄两性花、‘1613C'葡萄雄花和‘520A'葡萄雌花为材料，测定始蕾期、大蕾期和盛花期花器官的激素含量并进行转录组测序。各激素在不同性别花中的积累量不同，同一性别花器官不同发育时期激素含量也有差异，在大蕾期雌花中IAA、ABA、MeJA含量最高。转录和代谢联合分析共获得31 965个表达基因，其中与花发育直接相关的基因1 535个，部分基因持续上调或下调，持续上调的基因主要参与苯丙烷合成、植物激素信号转导等通路，持续下调基因主要参与淀粉和糖代谢、氨基酸合成等通路。AP1、SEP1、AG2、SOC1等MADS-box基因在不同花器官和不同发育时期差异表达。MADS-box基因、开花诱导整合子基因及解毒和应激相关基因的差异表达对花性别分化具有重要作用。

关键词: 葡萄, 花器官, RNA-seq, 内源激素, 花性别分化

Abstract:

In the current study，the flowers of three grape varieties‘Beichun'（hermaphrodite），‘1613C'（male） and‘520A'（female）were chosen. Hormone determination and transcriptome sequencing were performed at the early bud stage，large bud stage and blooming stage. The accumulation of various plant hormones in flowers of different genders was different，and so was that in flowers of the same gender at different developmental stages. The contents of IAA，ABA and MeJA were the highest in the large bud stage of female flowers. A total of 31 965 expressed genes were obtained，and 1 535 genes were directly related to flower development and were mainly involved in phenylpropane synthesis and plant hormone signal transduction pathways. Some genes are continuously up-regulated or down-regulated at different developmental stages. The genes that are continuously up-regulated are mainly involved in Phenylpropane synthesis，plant hormone signal transduction and other pathways. The genes that are continuously down-regulated are mainly involved in starch and sugar metabolism，and amino acid synthesis. MADS-box genes，such as AP1，SEP1，AG2 and SOC1 were significantly differently expressed in different floral organs at developmental stages. During flower sex differentiation，the differential expression of the MADS-box gene，the flower-induced integron gene，and the detoxification and stress-related genes play important roles.

Key words: grape, flower organ, RNA-seq, endogenous hormone, flower sex differentiation

韩凯, 罗尧幸, 焦晓博, 闫钊, Naomi Abe-Kanoh, 马小河, 纪薇. 葡萄不同性别花器官发育机制初探[J]. 园艺学报, 2023, 50(12): 2551-2567.

HAN Kai, LUO Yaoxing, JIAO Xiaobo, YAN Zhao, NAOMI Abe-Kanoh, MA Xiaohe, JI Wei. Preliminary Study on the Mechanism of Flower Organ Development and Sex Formation in Different Grapes[J]. Acta Horticulturae Sinica, 2023, 50(12): 2551-2567.

图/表 14

图1 葡萄3种性别花器官发育动态 S1、S2和S3分别表示始蕾期、大蕾期和盛花期。下同。

Fig. 1 Dynamic development of three sexual organs of grape S1，S2，and S3 represent early bud stage，large bud stage and blooming stage，respectively. The same below.

图2 葡萄3种性别花粉形态电镜观察

Fig. 2 Observation of different sex grapes pollen morphology by electron microscopy

图3 葡萄3种性别花器官发育过程中内源激素的变化不同字母表示相同时期样品间差异显著（LSD法，P ≤ 0.05）。

Fig. 3 Endogenous hormone content in three sexual organs of grape The different letters are significantly different between samples（LSD，P ≤ 0.05）.

表1 18个样品转录组测序基本信息

Table 1 Reads statistics based on RNA-Seq data of 18 libraries

样品 Sample	测序数量 Clean reads	基数 Clean bases	GC含量/% GC content	Q30/%	映射数量 Mapped reads	唯一映射数量 Unigene mapped reads	多重映射数量 Multiple mapped reads
FS1-1	35 072 390	10 460 784 892	46.92	89.21	21 544 969 (61.43%)	21 151 539 (60.31%)	394 306 (1.12%)
FS1-2	33 289 127	9 934 504 758	46.41	89.08	20 990 124 (63.06%)	20 598 256 (61.88%)	393 287 (1.18%)
FS2-1	31 912 591	9 508 367 080	47.16	90.12	18 860 341 (59.10%)	18 486 007 (57.93%)	373 997 (1.17%)
FS2-2	24 143 185	7 189 409 044	46.54	91.20	15 456 467 (64.02%)	15 134 525 (62.69%)	321 559 (1.33%)
FS3-1	34 577 903	10 309 343 968	46.29	89.46	21 528 202 (62.26%)	21 096 785 (61.01%)	432 896 (1.25%)
FS3-2	27 251 953	8 127 212 828	46.64	89.59	16 645 493 (61.08%)	16 324 467 (59.90%)	320 618 (1.18%)
MS1-1	34 721 924	10 360 502 876	46.44	93.05	21 968 561 (63.27%)	21 530 344 (62.01%)	438 215 (1.26%)
MS1-2	35 750 397	10 661 711 784	46.13	92.69	22 837 354 (63.88%)	22 373 027 (62.58%)	462 573 (1.29%)
MS2-1	31 178 630	9 301 801 016	47.21	92.29	18 516 988 (59.39%)	18 086 723 (58.01%)	429 967 (1.38%)
MS2-2	31 213 860	9 291 852 870	46.68	93.17	17 701 380 (56.71%)	18 898 728 (60.55%)	439 769 (1.41%)
MS3-1	34 905 725	10 394 750 300	46.69	89.27	20 032 396 (57.39%)	19 597 741 (56.14%)	434 693 (1.25%)
MS3-2	38 482 329	11 487 839 784	46.83	88.74	21 823 329 (56.71%)	21 337 468 (55.45%)	487 457 (1.27%)
HS1-1	34 651 419	10 338 334 932	46.33	89.44	23 854 037 (68.84%)	23 413 262 (67.57%)	441 714 (1.27%)
HS1-2	32 555 738	9 699 684 606	46.24	89.74	22 675 072 (69.65%)	22 257 892 (68.37%)	415 710 (1.28%)
HS2-1	37 521 061	11 191 155 650	46.40	89.56	25 413 015 (67.73%)	24 914 342 (66.40%)	498 766 (1.33%)
HS2-2	36 299 424	10 830 928 788	46.63	89.57	23 993 919 (66.10%)	23 533 171 (64.83%)	460 084 (1.27%)
HS3-1	22 028 667	6 563 311 378	46.32	89.03	15 005 928 (68.12%)	14 726 502 (66.85%)	279 417 (1.27%)
HS3-2	26 015 766	7 755 108 802	46.26	89.15	17 789 581 (68.38%)	17 444 441 (67.05%)	344 719 (1.33%)

表1 18个样品转录组测序基本信息

Table 1 Reads statistics based on RNA-Seq data of 18 libraries

样品 Sample	测序数量 Clean reads	基数 Clean bases	GC含量/% GC content	Q30/%	映射数量 Mapped reads	唯一映射数量 Unigene mapped reads	多重映射数量 Multiple mapped reads
FS1-1	35 072 390	10 460 784 892	46.92	89.21	21 544 969 (61.43%)	21 151 539 (60.31%)	394 306 (1.12%)
FS1-2	33 289 127	9 934 504 758	46.41	89.08	20 990 124 (63.06%)	20 598 256 (61.88%)	393 287 (1.18%)
FS2-1	31 912 591	9 508 367 080	47.16	90.12	18 860 341 (59.10%)	18 486 007 (57.93%)	373 997 (1.17%)
FS2-2	24 143 185	7 189 409 044	46.54	91.20	15 456 467 (64.02%)	15 134 525 (62.69%)	321 559 (1.33%)
FS3-1	34 577 903	10 309 343 968	46.29	89.46	21 528 202 (62.26%)	21 096 785 (61.01%)	432 896 (1.25%)
FS3-2	27 251 953	8 127 212 828	46.64	89.59	16 645 493 (61.08%)	16 324 467 (59.90%)	320 618 (1.18%)
MS1-1	34 721 924	10 360 502 876	46.44	93.05	21 968 561 (63.27%)	21 530 344 (62.01%)	438 215 (1.26%)
MS1-2	35 750 397	10 661 711 784	46.13	92.69	22 837 354 (63.88%)	22 373 027 (62.58%)	462 573 (1.29%)
MS2-1	31 178 630	9 301 801 016	47.21	92.29	18 516 988 (59.39%)	18 086 723 (58.01%)	429 967 (1.38%)
MS2-2	31 213 860	9 291 852 870	46.68	93.17	17 701 380 (56.71%)	18 898 728 (60.55%)	439 769 (1.41%)
MS3-1	34 905 725	10 394 750 300	46.69	89.27	20 032 396 (57.39%)	19 597 741 (56.14%)	434 693 (1.25%)
MS3-2	38 482 329	11 487 839 784	46.83	88.74	21 823 329 (56.71%)	21 337 468 (55.45%)	487 457 (1.27%)
HS1-1	34 651 419	10 338 334 932	46.33	89.44	23 854 037 (68.84%)	23 413 262 (67.57%)	441 714 (1.27%)
HS1-2	32 555 738	9 699 684 606	46.24	89.74	22 675 072 (69.65%)	22 257 892 (68.37%)	415 710 (1.28%)
HS2-1	37 521 061	11 191 155 650	46.40	89.56	25 413 015 (67.73%)	24 914 342 (66.40%)	498 766 (1.33%)
HS2-2	36 299 424	10 830 928 788	46.63	89.57	23 993 919 (66.10%)	23 533 171 (64.83%)	460 084 (1.27%)
HS3-1	22 028 667	6 563 311 378	46.32	89.03	15 005 928 (68.12%)	14 726 502 (66.85%)	279 417 (1.27%)
HS3-2	26 015 766	7 755 108 802	46.26	89.15	17 789 581 (68.38%)	17 444 441 (67.05%)	344 719 (1.33%)

图4 葡萄3种性别花器官在不同发育时期差异基因统计

Fig. 4 Statistics of differentially expressed gene numbers among different stages in three sexual organs of grape

图5 葡萄3种性别花器官MADS-box基因表达模式热图

Fig. 5 Heatmap of MADS-box gene expression patterns in three sexual organs of grape

图6 葡萄同一时期三3种性别葡萄花器官差异基因数统计 F、H和M分别表示雌花、两性花和雄花。

Fig. 6 Statistics of differentially expressed gene numbers among the three types of floral organ at the same stage F，H，and M represent female，hermaphrodite and male.

图7 葡萄3种性别花差异表达基因生物过程GO富集（前10项）右侧数字表示显著富集的基因数。

Fig. 7 GO（biological process）enrichment of differentially expressed genes involved in three types of grape flower development at the same stage（top 10） The numbers on the right indicate the significantly enriched genes.

图8 葡萄3个发育时期雌花（F）、两性花（H）和雄花（M）参与花器官形成的差异基因聚类热图所示的差异基因参与形成萼片、花瓣、雄蕊、雌蕊和心皮。

Fig. 8 Hierarchical clustering tree of differentially expressed genes involved in floral organ forming at three development stages in female（F），hermaphrodite（H）and male（M） Genes presented are involved in the formation of sepals，petal，stamen，gynoecium and carpel.

图9 葡萄3种性别花器官始蕾期（S1）、大蕾期（S2）和盛花期（S3）开花途径信号相关差异基因聚类热图所示的差异基因参与光周期途径、春化途径、自主途径和植物激素信号传导。

Fig. 9 Hierarchical clustering tree of differentially expressed genes involved in the flowering pathways of grape flower with three sexes in early bud stage（S1），large bud stage（S2）and blooming stage（S3） Genes shown are involved in photoperiod pathway，vernalization pathway，autonomuspathway，and plant hormone signal transduction pathways.

图10 雌、雄葡萄花差异基因韦恩图

Fig. 10 Venn dendragrams of differentially expressed genes between male and female flowers

表2 葡萄单性花发育过程中与激素相关的上下调基因数

Table 2 Number of hormone-related genes that are up- and down-regulated in unisexual flowers

激素 Hormones	始蕾期 Early bud stage		大蕾期 Large bud stage		盛花期Blooming stage
激素 Hormones	上调Up	下调Down	上调Up	下调Down	上调Up	下调Down
Auxin	116	150	149	193	204	233
GA	46	44	50	54	86	72
ZT	15	11	16	14	14	19
ABA	156	187	191	258	232	336
BR	54	81	82	89	121	107

表3 单性花中激素相关的上、下调基因

Table 3 Hormone-related genes that are up- and down-regulated in unisexual flowers

基因ID Gene ID	说明 Description	log₂FC
基因ID Gene ID	说明 Description	始蕾期 Early bud stage	大蕾期 Large bud stage	盛花期 Blooming stage
VIT_00s1203g00010	类生长素反应因子6 Auxin response factor 6-like	7.45	6.59	7.57
VIT_13s0019g01160	类黄烷酮4-还原酶Flavanone 4-reductase-like	3.32	3.60	5.68
VIT_11s0016g02110	细胞分裂素脱氢酶3 Cytokinin dehydrogenase 3	2.56	6.21	6.26
VIT_16s0098g01080	生长调节因子2 Growth-regulating factor 2	1.17	2.73	1.86
VIT_16s0022g02310	赤霉素氧化酶20 Gibberellin 20-oxidase	1.02	2.37	5.61
VIT_00s0634g00030	LRR受体丝氨酸/苏氨酸蛋白激酶 LRR receptor-like serine/threonine-protein kinase	-1.72	-1.94	-3.15
VIT_17s0000g09790	BTB/POZ域;TAZ锌指 BTB/POZ domain;TAZ zinc finger	-1.92	-1.85	-1.83
VIT_06s0004g00020	含有NAC结构域的蛋白质68 NAC domain-containing protein 68	-2.50	-3.10	-3.64
VIT_11s0052g00870	生长素响应蛋白IAA33 Auxin-responsive protein IAA33	-3.20	-5.68	-6.74
VIT_00s0324g00100	UDP糖基转移酶85A2 UDP-glycosyltransferase 85A2	-4.63	-7.99	-6.91

图11 葡萄花发育与性别分化相关基因qPCR验证

Fig. 11 qPCR validation of genes involved in grape flower development and sex specification

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