油橄榄四倍体构建及巨大化关键基因EXP家族鉴定及表达分析

doi:10.11707/j.1001-7488.LYKX20250570

摘要/Abstract

摘要：

目的: 构建油橄榄四倍体植株，分析其与野生型二倍体植株的表型差异，探索油橄榄四倍体巨大化表型形成的基因调控机制。方法: 使用秋水仙素和安磺灵混合溶液，以滴液法诱导植株，应用流式细胞术快速鉴定植株倍性；通过改良苯酚品红溶液染色，检测植株核型；通过振荡切片和甲苯胺蓝溶液染色，观察四倍体植株叶片横切面；通过PI染色，观察四倍体植株叶片表皮细胞；使用Illumina平台对不同倍性的叶片进行基因表达分析和DEGs富集分析；基于T2T油橄榄高质量基因组，利用Blastp、HMM、ProtParam、cello version 2.5、MEGA11、AlphaFold3.0、SAVES、PyMOL等分析平台，分析鉴定EXP基因家族成员，解析其基因表达模式。结果: 共筛选出23株四倍体油橄榄植株，得率为24.46%；四倍体与二倍体相比，植株出现巨大化的表型，表皮细胞和气孔极显著增大（P<0.0001）；共筛选到4 743个差异基因，其中2 371个上调，2 372个下调；通过差异基因分析，富集到与细胞巨大化密切相关的生长素信号转导通路，鉴定出2个显著上调的EXP成员，并进行qRT-PCR验证；在全基因组水平上完成了对EXP基因家族的分析。结论: EXP基因家族与四倍体细胞巨大化密切相关，为今后进一步探索多倍体巨大化表型奠定了基础。

关键词: 油橄榄, 四倍体, 巨大化, 转录组, EXP基因家族

Abstract:

Objective: This study aims to generate tetraploid olive plants, analyze their phenotypic differences from wild-type diploids, and explore the genetic regulatory mechanism underlying the cell enlargement phenotype in the tetraploids. Method: Tetraploid induction was performed using a colchicine and oryzalin mixture via a droplet application. Ploidy was rapidly identified by flow cytometry. Karyotype analysis was performed using improved phenol fuchsin staining. Leaf cross-sections of tetraploid plants were made using vibratome sectioning, and observed after toluidine blue staining. Leaf epidermal cells of tetraploids were examined by PI staining. Gene expression profiling and differentially expressed genes (DEGs) enrichment analysis of leaves of different ploidy plants were conducted using the Illumina platform. Based on the T2T high-quality olive genome, EXP gene family members were identified and characterized using Blastp, HMM, ProtParam, Cello version 2.5, MEGA11, AlphaFold3.0, SAVES, and PyMOL, and their expression patterns were further elucidated. Result: A total of 23 tetraploid olive plants were obtained, with an induction efficiency of 24.46%. Compared to diploids, tetraploids exhibited a cell enlargement phenotype, with significantly enlarged epidermal cells and stomata (P<0.000 1). RNA-seq identified 4 743 DEGs (2 371 up-regulated and 2 372 down-regulated). Through differential gene expression analysis, the auxin signaling pathway closely associated with cell enlargement was significantly enriched. Two markedly upregulated EXP members were identified and further validated by qRT-PCR. A comprehensive genome-wide analysis of the EXP gene family was completed. Conclusion: The EXP gene family is closely associated with tetraploid cell enlargement, laying a foundation for further exploration of the gigas phenotype in polyploids in the future.

Key words: Olea europaea, polyploid, cell enlargement, transcriptome, EXP gene family

中图分类号:

S722.3⁺5

李雨霏,易刚,李书媛,崔祺禛,樊育彤,饶国栋. 油橄榄四倍体构建及巨大化关键基因EXP家族鉴定及表达分析[J]. 林业科学, 2026, 62(6): 166-177.

Yufei Li,Gang Yi,Shuyuan Li,Qizhen Cui,Yutong Fan,Guodong Rao. Construction of Tetraploid Olive (Olea europaea) and Identification and Expression Analysis of Key Genes in the EXP Family for the Cell Enlargement Phenotype[J]. Scientia Silvae Sinicae, 2026, 62(6): 166-177.

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基因名称 Gene name	引物序列（5’–3’） Primer sequence （5’–3’）
Lec47749	F: TACTCATAACCAATGTCGGGGAG	R: ATTGTTAGAAACCATTGTACGGCC
Lec09513	F: AATTACCCTGGAGTACGATTGACC	R: CTTCCCAGAATCCCCAGTTATCTT
Tubulin	F: CGTTCATTGGAAACTCAACCTCAA	R: TTCTTCCTCGTATTCCTCATCGTC

Seq_1	Seq_2	Ka	Ks	Ka/Ks
OeEXPA1	OeEXPA11	0.102	2.237	0.045
OeEXPA25	OeEXPA17	0.116	2.274	0.051
OeEXPA1	OeEXPA26	0.107	1.986	0.054
OeEXPA17	OeEXPA34	0.115	2.004	0.057
OeEXPA25	OeEXPA34	0.143	2.386	0.060
OeEXPA1	OeEXPA19	0.099	1.615	0.061
OeEXPA16	OeEXPA1	0.161	2.615	0.062
OeEXPA16	OeEXPA26	0.148	2.001	0.074
OeEXPA12	OeEXPA27	0.026	0.349	0.075
OeEXPA1	OeEXPA22	0.104	1.265	0.082
OeEXPA22	OeEXPA19	0.031	0.374	0.082
OeEXPA7	OeEXPA12	0.069	0.799	0.086
OeEXPA21	OeEXPA20	0.083	0.954	0.086
OeEXPA19	OeEXPA26	0.070	0.801	0.087
OeEXPA3	OeEXPA25	0.083	0.893	0.093
OeEXPA22	OeEXPA26	0.076	0.817	0.094
OeEXPA11	OeEXPA26	0.033	0.330	0.101
OeEXPA24	OeEXPA20	0.040	0.399	0.101
OeEXPA16	OeEXPA11	0.157	1.509	0.104
OeEXPA8	OeEXPA18	0.042	0.394	0.106
OeEXPA3	OeEXPA28	0.102	0.941	0.108
OeEXPA11	OeEXPA22	0.079	0.727	0.109
OeEXPA15	OeEXPA13	0.097	0.828	0.118
OeEXPA2	OeEXPA13	0.100	0.852	0.118
OeEXPA11	OeEXPA19	0.074	0.594	0.125
OeEXPA25	OeEXPA28	0.050	0.381	0.130
OeEXPA2	OeEXPA5	0.107	0.821	0.130
OeEXPA15	OeEXPA2	0.038	0.290	0.131
OeEXPA15	OeEXPA5	0.100	0.675	0.148
OeEXPA10	OeEXPA34	0.048	0.278	0.174
OeEXPA14	OeEXPA26	0.227	1.241	0.183
OeEXPA5	OeEXPA13	0.040	0.200	0.200
OeEXPA10	OeEXPA23	0.331	1.631	0.203
OeEXPA14	OeEXPA11	0.223	1.069	0.209
OeEXPA14	OeEXPA1	0.119	0.496	0.240
OeEXPB2	OeEXPB4	0.456	1.494	0.305
OeEXPA3	OeEXPA30	0.113	0.319	0.356
OeEXPB2	OeEXPB9	0.484	1.121	0.432
OeEXPB4	OeEXPB9	0.219	0.469	0.467
OeEXLB3	OeEXLB7	0.276	0.570	0.484
OeEXLB16	OeEXLB17	0.302	0.625	0.484
OeEXLB5	OeEXLB6	0.178	0.362	0.492
OeEXLB17	OeEXLB18	0.179	0.307	0.583
OeEXLB11	OeEXLB12	0.071	0.110	0.642
OeEXLB18	OeEXLB19	0.154	0.237	0.648
OeEXLB9	OeEXLB10	0.492	0.746	0.660
OeEXLB13	OeEXLB14	0.045	0.046	0.968
OeEXLB19	OeEXLB20	0.024	0.020	1.200

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