低温胁迫下枇杷不同发育阶段的花果转录组比较分析

doi:10.16420/j.issn.0513-353x.2021- 0219

摘要/Abstract

摘要：

研究比较了枇杷（Eriobotrya japonica Lindl.）不同发育阶段花果的低温响应机制。以花蕾、完全开放的花和花后2周左右的幼果为试验材料,-3 ℃低温处理12 h,以未经低温处理样品为对照,测定生理生化指标并进行转录组测序分析。低温胁迫导致细胞膜破坏,超氧阴离子产生速率、丙二醛和脯氨酸含量、抗氧化酶活性明显升高。总体上抗低温能力为花蕾 > 花 > 幼果。转录组测序分析共获得6 987个差异表达基因（DEG）,对这些基因进行通路注释分析,发现大量与低温胁迫相关的代谢途径,其中碳水化合物代谢中的糖酵解/糖异生、乙醛酸和二羧酸代谢及磷酸肌醇代谢途径,氨基酸代谢中的色氨酸代谢和酪氨酸代谢途径,酯类代谢中的甘油磷脂代谢、α-亚麻酸代谢和甘油酯代谢途径,次生代谢中的异喹啉生物碱生物合成途径以及能量代谢中的硫代谢途径在3个花果发育时期低温与对照的比较组中显著富集,说明这几个途径都是枇杷花果响应低温胁迫的重要代谢途径。“苯丙烷类生物合成”途径在前两个时期比较组中富集水平较高,氨基酸代谢相关途径在幼果比较组中富集更多。另外,从低温处理相关差异表达基因中筛选到了53个AP2-EREBP基因,14个WRKY基因和15个NAC基因,并对其中12个低温响应相关转录因子基因进行了qRT-PCR表达分析,进一步证实了转录组数据的准确性。

关键词: 枇杷, 花, 果实, 发育阶段, 低温, 转录组, 差异基因, 转录因子

Abstract:

The study compared the low temperature response mechanism of loquat（Eriobotrya japonica Lindl.）flowers and fruits at different developmental stages. Flower buds,fully open flowers and young fruits were used as the experimental materials,treated at-3 ℃ for 12 h,and samples without low temperature treatment were used as controls. The relevant physiological and biochemical indexes,as well as transcriptome were determined both in control and treated samples. Low temperature stress led to the destruction of cell membranes,the generation rate of superoxide anion,the content of malondialdehyde and proline,and the activity of antioxidant enzymes were dramatically increased. In general,the low temperature resistance ability was flower bud > flower > young fruit. Transcriptome sequencing analysis obtained a total of 6 987 differentially expressed genes（DEG）. A large number of metabolic pathways related to low temperature stress were found,including glycolysis/gluconeogenesis,glyoxylic acid and dicarboxylic acid metabolism and phosphoinositol metabolism pathways in carbohydrate metabolism,tryptophan metabolism and tyrosine metabolism pathways in amino acid metabolism,glycerol phospholipid metabolism,α-linolenic acid metabolism and glyceride metabolism pathways in ester metabolism,isoquinoline alkaloid biosynthesis pathway in secondary metabolism,as well as thiometabolism pathway in energy metabolism,were significantly enriched in all comparison groups of low temperature and control at three flower and fruit development stages,which indicated that these pathways were important metabolic pathways in response to low temperature stress in loquat. The phenylpropanoid biosynthesis pathway was significantly enriched in both flower bud and flower comparison groups,and more amino acid metabolism pathways were enriched in young fruit comparison group after low temperature treatment. In addition,53 AP2-EREBP genes,14 WRKY genes and 15 NAC genes were screened from the differentially expressed genes related to low temperature treatment,and 12 low temperature response-related transcription factor genes were analyzed by qRT-PCR,which further confirmed the accuracy of transcriptome data.

Key words: Eriobotrya japonica, flower, fruit, development stage, low temperature, transcriptome, differentially expressed gene, transcription factor

中图分类号:

S667.3

徐红霞, 周慧芬, 李晓颖, 姜路花, 陈俊伟. 低温胁迫下枇杷不同发育阶段的花果转录组比较分析[J]. 园艺学报, 2021, 48(9): 1680-1694.

XU Hongxia, ZHOU Huifen, LI Xiaoying, JIANG Luhua, CHEN Junwei. Comparative Transcriptome Analysis of Different Developmental Stages of Flowers and Fruits in Loquat Under Low Temperature Stress[J]. Acta Horticulturae Sinica, 2021, 48(9): 1680-1694.

图/表 11

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基因名称 Gene name	基因 ID Gene ID	引物序列（5′-3′） Primer sequence
Ejactin	c158550.graph_c1	F：GGATTTGCTGGTGATGATGC;	R：CCGTGCTCAATGGGATACTT
ERF37	c106174.graph_c0	F：GCTGGGTGAGGAGATAACGG	R：AAGATGCGAAGACGGTGGTG
ERF73	c123742.graph_c0	F：GCTTTCCTGAGGCGGACGAT	R：AATTGAGCTTGGAGGCGATT
ERF35	c125424.graph_c0	F：GGGGAGAGCATAGCGATACA	R：GCTTACGTCATTTTCTGGGC
ERF105	c126492.graph_c0	F：TCGCTCGTACTTGTCACACC	R：GCTGACCATCAAAGGCAACT
RAV2	c126935.graph_c1	F：GAGGATGATCGGCTGGGAGT	R：AGCTGCTGCAAGGCACTACA
ERF60	c129543.graph_c0	F：TGGCCAATAAGAATCACCAA	R：CCAAAAAGGATCTGACGAGG
ERF33	c46214.graph_c0	F：GCCTGTTCTTTGATTTTGGT	R：TCATGAGCTGATCTTCCCTC
WRKY33	c142932.graph_c0	F：CCACTGAAAACCAATCGTAC	R：CTTAGACTTCTTATCGCAAC
NAC72	c51471.graph_c0	F：ATGTTGGGGTTGGATAAGAA	R：CGGAATAGAAGAAGCAGAAGAG
NAC74	c136256.graph_c0	F：AGTCCATAGTATGCGCCACC	R：TGACTGCGGTTTTCTTTTGA
NAC29	c130635.graph_c0	F：GAAACGGGAACATTGGTAGT	R：AATGAATGAGATGCTGGTGT
NAC91	c136368.graph_c0	F：ATCATCGCTAAATTCATTCC	R：ACTTTACCACCTCTACCAAC

基因名称 Gene name	基因 ID Gene ID	引物序列（5′-3′） Primer sequence
Ejactin	c158550.graph_c1	F：GGATTTGCTGGTGATGATGC;	R：CCGTGCTCAATGGGATACTT
ERF37	c106174.graph_c0	F：GCTGGGTGAGGAGATAACGG	R：AAGATGCGAAGACGGTGGTG
ERF73	c123742.graph_c0	F：GCTTTCCTGAGGCGGACGAT	R：AATTGAGCTTGGAGGCGATT
ERF35	c125424.graph_c0	F：GGGGAGAGCATAGCGATACA	R：GCTTACGTCATTTTCTGGGC
ERF105	c126492.graph_c0	F：TCGCTCGTACTTGTCACACC	R：GCTGACCATCAAAGGCAACT
RAV2	c126935.graph_c1	F：GAGGATGATCGGCTGGGAGT	R：AGCTGCTGCAAGGCACTACA
ERF60	c129543.graph_c0	F：TGGCCAATAAGAATCACCAA	R：CCAAAAAGGATCTGACGAGG
ERF33	c46214.graph_c0	F：GCCTGTTCTTTGATTTTGGT	R：TCATGAGCTGATCTTCCCTC
WRKY33	c142932.graph_c0	F：CCACTGAAAACCAATCGTAC	R：CTTAGACTTCTTATCGCAAC
NAC72	c51471.graph_c0	F：ATGTTGGGGTTGGATAAGAA	R：CGGAATAGAAGAAGCAGAAGAG
NAC74	c136256.graph_c0	F：AGTCCATAGTATGCGCCACC	R：TGACTGCGGTTTTCTTTTGA
NAC29	c130635.graph_c0	F：GAAACGGGAACATTGGTAGT	R：AATGAATGAGATGCTGGTGT
NAC91	c136368.graph_c0	F：ATCATCGCTAAATTCATTCC	R：ACTTTACCACCTCTACCAAC

数据库 Database	注释数量 Number of annotated unigene	比例/% Percentage of annotated unigene
Nr	38 093	97.80
Swiss-Prot	22 119	56.79
KEGG	14 813	38.03
eggNOG	35 024	89.92
KOG	20 989	53.89
GO	25 436	65.30
Pfam	27 156	69.72

数据库 Database	注释数量 Number of annotated unigene	比例/% Percentage of annotated unigene
Nr	38 093	97.80
Swiss-Prot	22 119	56.79
KEGG	14 813	38.03
eggNOG	35 024	89.92
KOG	20 989	53.89
GO	25 436	65.30
Pfam	27 156	69.72

GO分类 GO classification	GO序列号 GO accession	注释功能（词条） Term	差异基因数 DEGs number	-3 ℃ vs 25 ℃
GO分类 GO classification	GO序列号 GO accession	注释功能（词条） Term	差异基因数 DEGs number	花蕾 Flower bud	花 Flower	幼果 Young fruit
生物学过程 Biological process	GO:0008152	代谢过程Metabolic process	2 193	298	781	1 239
	GO:0009987	细胞过程Cellular process	2 127	248	661	1 075
	GO:0044699	单一生物过程Single-organism process	1 863	230	585	1 033
	GO:0051179	定位Localization	790	108	276	546
	GO:0065007	生物调控Biological regulation	616	142	270	427
	GO:0071840	细胞组分或生物合成 Cellular component organization or biogenesis	465	40	132	293
	GO:0050896	应激反应Response to stimulus	457	94	193	279
	GO:0023052	信号Signaling	122	24	47	77
	GO:0051704	多生物过程Multi-organism process	114	11	39	73
	GO:0032502	发育过程Developmental process	94	25	35	46
	GO:0040007	生长Growth	75	2	16	41
	GO:0022414	生殖过程Reproductive process	59	15	25	44
	GO:0000003	再生Reproduction	55	0	26	46
	GO:0032501	多细胞生物过程Multicellular organismal process	48	17	22	17
	GO:0022610	生物粘附Biological adhesion	17	0	3	6
	GO:0002376	免疫系统过程Immune system process	7	0	2	0
	GO:0048511	节律过程Rhythmic process	3	0	1	1
细胞组分 Cellular component	GO:0005623	细胞Cell	1 808	283	718	1 229
	GO:0044464	细胞部分Cell part	1 796	280	710	1 222
	GO:0043226	细胞器Organelle	1 260	201	485	858
	GO:0016020	膜Membrane	1 642	294	623	1 024
	GO:0044425	膜部分Membrane part	1 293	234	495	805
	GO:0044422	细胞器部分Organelle part	621	67	218	418
	GO:0032991	大分子复合物Macromolecular complex	552	42	208	361
	GO:0031974	膜封闭的管腔Membrane-enclosed lumen	160	11	40	112
	GO:0005576	细胞外区域Extracellular region	87	18	45	61
	GO:0030054	细胞连接Cell junction	15	4	6	5
	GO:0055044	共质体Symplast	14	4	5	5
	GO:0019012	病毒Virion	4	0	1	4
	GO:0044423	病毒部分Virion part	4	0	1	4
	GO:0009295	内核Nucleoid	11	0	5	6
	GO:0044421	细胞外区域Extracellular region part	28	3	21	26
分子功能	GO:0003824	催化活性Catalytic activity	1 949	308	725	1 222
Molecular	GO:0005488	联结Binding	1 670	321	688	1 024
function	GO:0005215	转运活动Transporter activity	402	53	144	277
	GO:0005198	结构分子活动Structural molecule activity	178	9	102	85
	GO:0001071	核酸联结转录因子活性 Nucleic acid binding transcription factor activity	109	40	64	68
	GO:0016209	抗氧化活性Antioxidant activity	40	10	21	21
	GO:0009055	电子载体活动Electron carrier activity	35	5	13	20
	GO:0060089	分子传感器活动Molecular transducer activity	36	5	7	8
	GO:0004871	信号传导活动Signal transducer activity	36	10	18	23
	GO:0000988	转录因子活性,蛋白质结合 Transcription factor activity,protein binding	16	4	3	12
	GO:0045735	营养库活性Nutrient reservoir activity	5	1	3	2
	GO:0031386	蛋白质标签Protein tag	5	0	0	2
	GO:0016530	金属伴侣活性Metallochaperone activity	1	1	1	1
	GO:0045182	翻译调控活性Translation regulator activity	2	0	0	2