长期继代培养的楸树胚性愈伤组织难以分化的成因

doi:10.11707/j.1001-7488.LYKX20240340

摘要/Abstract

摘要：

目的: 探究长期继代下楸树胚性愈伤组织难以分化再生的发生机制，为楸树体细胞胚胎发生体系的分化再生和遗传稳定性提供理论基础。方法: 以新诱导的非胚性愈伤组织和初代胚性愈伤组织、继代培养7年的胚性愈伤组织为材料，开展细胞倍性、差异表达基因及内源激素水平分析。结果: 1）通过染色体计数发现，初代胚性愈伤组织染色体保持二倍体状态（2n=40），而连续培养7年的胚性愈伤组织染色体数目发生了加倍现象，染色体数目为80~162；流式细胞仪鉴定发现连续培养 7 年的胚性愈伤 DNA 含量相比楸树实生苗增加 3 倍，其细胞倍性为六倍体。2）通过对转录组数据的GO和KEGG富集分析，发现差异表达基因显著富集在植物激素激活信号通路、转录调控、MAPK 信号通路、发育过程的调控等通路，其中响应生长素信号的SAUR、ARF、AUX/IAA、GH3，脱落酸受体PYR/PYL，胞外蛋白AGP，转录因子ABI3等促进体胚分化发生的基因表达均下调，而乙烯合成抑制因子ETO1则表达上调。3）内源激素水平分析表明，连续继代 7 年的胚性愈伤组织中IAA 和 ABA 的含量显著低于新诱导的胚性愈伤组织，这与其参与植物激素信号转导基因的下调表达相对应，而高水平的IAA和ABA可能是调控体细胞胚胎发生的关键。结论: 楸树胚性愈伤组织在长期继代过程中发生的染色体加倍、促进体胚发生相关基因的异常表达以及内源激素水平的显著下降，可能是造成其难以分化再生的根本原因。

关键词: 楸树, 胚性愈伤组织, 染色体加倍, 转录组测序, 内源激素

Abstract:

Objective: This study aims to explore the mechanism of difficulty in differentiation and regeneration of embryogenic callus of Catalpa bungei under long-term subculture, so as to provide a theoretical basis for the differentiation and regeneration of somatic embryogenesis system and genetic stability of C. bungei. Method: The newly induced non-embryogenic callus, primary embryogenic callus and 7-year subcultured embryogenic callus were used as materials to analyze the cell ploidy, differentially expressed genes and endogenous hormone levels. Result: 1) Through chromosome counting, it was found that the chromosome number of the primary embryogenic callus maintained diploid status (2n = 40), while the chromosome number of the embryogenic callus continuously cultured for 7 years was doubled, and the chromosome number ranged from 80 to 162. The result of flow cytometry measurement showed that the DNA content of embryogenic callus that was cultured continuously for 7 years was 3 times higher than that of C. bungei seedlings, and the cell ploidy was hexaploid. 2) Through GO and KEGG enrichment analysis of transcriptome data, it was found that differentially expressed genes were significantly enriched in plant hormone activation signaling pathway, transcriptional regulation, MAPK signaling pathway, development process regulation and other pathways. Among them, SAUR, ARF, AUX/IAA and GH3 which respond to auxin signaling, abscisic acid receptor PYR/PYL, extracellular protein AGP, transcription factor ABI3 and other genes that promote somatic embryo differentiation were all down-regulated, while the expression of ethylene synthesis inhibitor ETO1 was up-regulated. 3) Endogenous hormone level analysis showed that the content of IAA and ABA in embryogenic callus that was cultured continuously for 7 years was significantly lower than that in newly induced embryogenic callus, which corresponded to the down-regulated expression of genes involved in plant hormone signal transduction. It is suggested that high levels of IAA and ABA may be the key to regulating somatic embryogenesis. Conclusion: In summary, it is speculated that chromosome doubling, abnormal expression of genes related to somatic embryogenesis and significant decrease of endogenous hormone levels in the embryogenic callus of C. bungei during long-term subculture may be the fundamental cause of its difficulty in differentiation and regeneration.

Key words: Catalpa bungei, embryogenic callus, chromosome doubling, transcriptome sequencing, endogenous hormones

中图分类号:

S718.46

赵珊,董静,郑帅,陈发菊,刘文,梁宏伟. 长期继代培养的楸树胚性愈伤组织难以分化的成因[J]. 林业科学, 2025, 61(6): 130-138.

Shan Zhao,Jing Dong,Shuai Zheng,Faju Chen,Wen Liu,Hongwei Liang. Reasons for the Difficulty of Differentiation of Embryogenic Callus in Long-Term Subculture of Catalpa bungei[J]. Scientia Silvae Sinicae, 2025, 61(6): 130-138.

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基因名称 Gene name	序列 Sequences
Cabuactin-F	5'-(GATGATGCTCCAAGAGCTGT)-3'
Cabuactin-R	5'-(TCCATATCATCCCAGTTGCT)-3'
CabuETO1-F	5'-(AGCAGATGAAACTCCAAGTTATGC)- 3'
CabuETO1-R	5'-(AGGGGAAATATCGTTTTCAGACAG)-3'
CabuPYR/PYL1-F	5'-(CTCCGTCACGACCTTACACTCC)-3'
CabuPYR/PYL1-R	5'-(GAGAGGTTTCGTGTGGCTAAATTC)-3'
CabuPYR/PYL2-F	5'-(CTACCGTTCCGTCACAACACTAC)-3'
CabuPYR/PYL2-R	5'-(AACTGACCATACGGTGGAGATG)-3'
CabuAGP-F	5'-(ACTCCCTCGCCACTATCAAAAAC)-3'
CabuAGP-R	5'-(AATCCGACCTTACCTCCTTTCATG)-3'
CabuARF-F	5'-(CGTGAACTAGATGACCATCAATCTCC)-3'
CabuARF-R	5'-(CCAAAAGACATCTTCGCAGTAATCC)-3'
CabuAUX/IAA-F	5'-(GGGTAATTATGGGAGCCAAGACATG)-3'
CabuAUX/IAA-R	5'-(CAAGTCCAATTGCTTCAGATCCTTTC)-3'

样本名称 Sample name	MFI 1	MFI 2	MFI 3	G0/G1 荧光均值 G0/G1 fluorescence mean value	倍性 Multiplicity
幼苗 Seedlings	421 383.7	409 580.4	421 357.3	417 440.5	2.0
愈伤组织 Callus	1 250 581.3	1 256 419.5	1 260 507.8	1 255 836.2	6.0

数据库 Databases	注释数量 Number	注释比例 Percentage (%)
NR	65 571	79.58
NT	59 604	72.34
Swissprot	50 009	60.69
KEGG	51 237	62.18
KOG	51 755	62.81
GO	54 048	65.60
Intersection	31 701	38.47
Overall	67 727	82.20
All Unigene	82 396	100

家族 Family	对照表达量 CK expression	处理表达量 Treat expression	log₂倍数变化 log₂ fold change	Q值 Q-value	P值 P-value
SAUR	8.48	1.84	–2.20	0.001466	4.29E-05
ARF	2.09	0.86	–1.31	0.025 401	0.001 532
	9.10	3.50	–1.42	0.006 374	0.000 259
	1.96	0.73	–1.45	0.000 573	1.39E-05
	33.12	15.33	–1.18	0.014 215	0.000 716
	4.90	0.06	–5.01	3.15E-08	1.97E-10
	6.67	4.29	–0.71	0.026 807	0.001 645
	1.57	0.04	–4.97	5.60E-11	2.02E-13
	5.04	3.00	–0.82	0.004 952	0.000 188
	1.91	0.74	–1.43	0.002 142	6.73E-05
	3.49	1.52	–1.23	0.040 654	0.002 924
	12.87	8.44	–0.70	0.028 643	0.001 803
AUX/IAA	68.94	25.85	–1.52	6.96E-08	4.76E-10
	21.94	4.72	–2.28	1.50E-08	8.91E-11
	4.09	0.28	–3.25	0.000 593	1.45E-05
	16.63	1.53	–3.05	0.000 158	2.98E-06
	26.83	2.15	–3.45	3.39E-09	1.76E-11
	12.20	0.44	–4.70	1.36E-23	7.91E-27
	8.06	2.42	–1.78	0.003 030	0.000 102
	5.88	2.10	–1.54	1.45E-05	1.93E-07
	14.68	7.90	–0.97	0.000 119	2.16E-06
	19.19	8.32	–1.27	0.014 830	0.000 755
	21.9	5.37	–1.96	0.003 016	0.000 102
GH3	16.73	11.64	–0.61	0.007 560	0.000 323
	16.74	6.81	–1.34	0.001 868	5.69E-05
	1.15	0.18	–2.28	0.040 221	0.002 877
AGP	14.84	9.89	–0.69	0.031 964	0.002 098
	4.97	3.50	–0.59	0.026 700	0.001 636
	48.42	8.70	–2.50	9.32E-13	2.36E-15
	4.56	0.78	–2.49	0.000 165	3.16E-06
	25.38	17.48	–0.63	0.011 500	0.000 545
	2.48	0.61	–1.93	0.021 617	0.001 244
	36.19	1.90	–3.87	8.69E-08	6.08E-10
ETO1	8.52	32.68	1.80	1.51E-06	1.45E-08
ABI3	9.16	4.13	–1.22	0.000 114	2.06E-06
	3.33	1.31	–1.38	0.022 468	0.001 307
	5.89	3.77	–0.72	0.030 067	0.001 928
	6.73	2.37	–1.57	0.007 703	0.000 331
PYR/PYL	23.83	8.56	–1.47	0.038 350	0.002 700
	35.06	12.35	–1.62	1.95E-11	6.25E-14
	43.40	16.27	–1.49	4.78E-05	7.45E-07
	5.60	0.87	–2.68	1.57E-07	1.19E-09

愈伤组织类型 Types of callus	IAA	ABA	CTK	GA₃
继代7年胚性愈伤 Successional 7-year embryonic callus	54.21±1.09c	177.67±4.77c	6.14±0.16c	0.31±0.003c
新诱导胚性愈伤 Newly induced embryonic callus	80.97±1.34a	300.67±9.29a	8.73±0.13a	0.47±0.008a
非胚性愈伤 Non-embryonic callus	64.50±0.79b	227.05±4.57b	6.64±0.23b	0.35±0.006b