茉莉花FLA基因家族鉴定及JsFLA2的功能分析

doi:10.3864/j.issn.0578-1752.2025.17.012

摘要/Abstract

摘要：

【目的】茉莉花的生殖障碍十分严重，种质创新工作长期受阻。类成束阿拉伯半乳糖蛋白（fasciclin-like arabinogalactan proteins，FLAs）在植物生殖发育过程中起着重要作用。通过对茉莉花FLA基因家族进行全基因组鉴定与分析，研究其基本特性及表达模式，为深入解析JsFLAs在茉莉花生殖发育中的生物学功能提供理论依据。【方法】利用HMMER 3.0软件在茉莉花全基因组中筛选鉴定FLA基因家族成员，并通过Smart和CD-Search进一步验证；利用ExPASy、SignalP 5.0、big-PI Plant Predictor、NetNGlyc-1.0和Plant-mPLoc等网站分析JsFLAs的理化性质及亚细胞定位；利用MEGA、TBtools、MEME、PlantCARE等对JsFLAs进行系统进化树构建、基因染色体定位、基因结构分析、蛋白保守基序分析及其启动子顺式作用元件预测；利用转录组数据分析JsFLAs在授粉后不同时间的柱头和不同发育阶段花中的表达特征；采用实时荧光定量PCR（qRT-PCR）检测JsFLA2在茉莉花不同组织中的表达特性，并通过瞬时转化烟草叶片试验分析其亚细胞定位；最后，通过异源转化拟南芥验证JsFLA2的生物学功能。【结果】在茉莉花全基因组中共鉴定出24个JsFLAs，命名为JsFLA1—JsFLA24，其蛋白包含159—515个氨基酸残基，分子量为17.02—56.34 kDa，等电点为4.20—9.69；所有成员均含有1—2个类成束蛋白结构域、1—2个AGP糖基化位点和1个糖基化磷脂酰肌醇锚定位点，其中19个成员含有信号肽；除JsFLA5位于细胞核外，其余成员均位于细胞膜；茉莉花FLAs蛋白可分为4个亚类，且蛋白保守基序在各组中相对保守；JsFLAs不均匀地分布在11条染色体上；JsFLAs含0—3个内含子及1—3个外显子；21个JsFLAs在授粉后不同时间的柱头表达，20个JsFLAs在不同发育阶段的花中表达；JsFLAs启动子含有生长发育元件、植物激素响应元件和非生物胁迫响应元件等。此外，具有显著差异表达的JsFLA2编码405个氨基酸，与油橄榄OeFLA1亲缘关系最近，在花中表达量最高，定位于细胞膜。异源转化拟南芥结果显示，转基因株系表现出花粉活力降低、种子数减少和角果变短等严重的结实障碍。【结论】在茉莉花中鉴定出24个FLA基因家族成员，明确了JsFLA2与茉莉花的花粉育性密切相关。

关键词: 茉莉花, FLA基因家族, 表达分析, JsFLA2, 功能分析

Abstract:

【Objective】Jasmine (Jasminum sambac) exhibits significant sexual reproductive barriers, severely impeding germplasm innovation. Fasciclin-like arabinogalactan proteins (FLAs) play crucial roles in plant reproduction development. This study conducted a genome-wide identification and analysis of the FLA gene family in jasmine, then investigated their fundamental characteristics and expression patterns, thereby providing a theoretical foundation for understanding the biological functions of the JsFLAs in reproduction development.【Method】The HMMER 3.0 software was used to screen and identify members of the FLA gene family in the jasmine genome, with further validation through Smart and CD-Search. Websites such as ExPASy, SignalP 5.0, big-PI Plant Predictor, NetNGlyc-1.0, and Plant-mPLoc were used to analyze the physicochemical properties and subcellular location of JsFLAs. MEGA, TBtools, MEME, and PlantCARE were utilized for phylogenetic tree construction, chromosome location, gene structure analysis, protein conserved motif identification, and promoter cis-element prediction. Transcriptional data were analyzed to determine the expression characteristics of JsFLAs in stigmas at various time points after pollination and in flowers at different developmental stages. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of JsFLA2 in jasmine, and its subcellular location was analyzed through transient transformation of tobacco leaves. Finally, the biological function of JsFLA2 was verified through heterologous expression in Arabidopsis thaliana.【Result】A total of 24 JsFLAs were identified in the jasmine genome, named JsFLA1-JsFLA24. These proteins consisted of 159-515 amino acid residues, with molecular weight of 17.02-56.34 kDa and isoelectric point of 4.20-9.69. All members contained 1-2 fasciclin-like domain (FAS), 1-2 AGP glycosylation sites, and a glycosylphosphatidylinositol (GPI) anchored site, and 19 members contained signal peptide (SP). Except for JsFLA5 which was localized in the nucleus, all other members were located on the plasma membrane. The JsFLAs were divided into four subclasses, with conserved motifs within each group. The JsFLAs were unevenly distributed across 11 chromosomes. The JsFLAs contained 0-3 introns and 1-3 exons. 21 JsFLAs were expressed in stigmas at various time points after pollination, and 20 were expressed in flowers at different developmental stages. The JsFLAs promoters included elements responsive to growth and development, plant hormones, and abiotic stress. Further research revealed that the significantly differentially expressed JsFLA2 encodes 405 amino acids, is most similar to OeFLA1, exhibits the highest expression in flowers, and is localized on the plasma membrane. Heterologous expression of JsFLA2 in A. thaliana showed that the transgenic lines exhibited severe fertility defects, including reduced pollen viability, decreased seed number, and shorter siliques.【Conclusion】This study identified 24 FLA gene family members in jasmine. Functional validation confirmed that JsFLA2 is closely related to pollen fertility in jasmine.

Key words: Jasminum sambac, FLA gene family, expression analysis, JsFLA2, functional analysis

齐香玉, 李新茹, 陈双双, 冯景, 陈慧杰, 刘欣童, 金玉妍, 邓衍明. 茉莉花FLA基因家族鉴定及JsFLA2的功能分析[J]. 中国农业科学, 2025, 58(17): 3516-3530.

QI XiangYu, LI XinRu, CHEN ShuangShuang, FENG Jing, CHEN HuiJie, LIU XinTong, JIN YuYan, DENG YanMing. Identification of the FLA Gene Family and Functional Analysis of JsFLA2 in Jasminum sambac[J]. Scientia Agricultura Sinica, 2025, 58(17): 3516-3530.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.17.012

https://www.chinaagrisci.com/CN/Y2025/V58/I17/3516

图/表 9

表1

茉莉花24个JsFLAs的基本信息"

基因 Gene	氨基酸数量 No. of amino acid (aa)	分子量 Molecular weight (kDa)	等电点Isoelectric point (pI)	FAS结构域位置Location of FAS domain (aa)	糖基化位点N-glycosylation sites (aa)	GPI锚定位点GPI anchor site (aa)	信号肽位点Signal peptide site (aa)	亚细胞定位 Subcellular location
JsFLA1	243	25.51	7.79	45—180	36, 140	218	22—23	细胞膜Plasma membrane
JsFLA2	405	42.91	5.46	23—128	23, 158	381	21—22	细胞膜Plasma membrane
JsFLA3	472	51.77	6.07	321—426	88	441	27—28	细胞膜Plasma membrane
JsFLA4	460	50.56	6.08	316—421	40, 83	431	26—27	细胞膜Plasma membrane
JsFLA5	451	48.65	4.20	50—148	125, 352	427	29—30	细胞核Nucleus
JsFLA6	429	44.12	5.14	200—331	30, 131	402	25—26	细胞膜Plasma membrane
JsFLA7	349	37.57	6.66	242—311	81	337	28—29	细胞膜Plasma membrane
JsFLA8	286	31.33	5.55	76—179	118	265	20—21	细胞膜Plasma membrane
JsFLA9	286	31.22	5.77	76—179	70, 118	265	20—21	细胞膜Plasma membrane
JsFLA10	204	22.44	5.70	79—185	97	167	—	细胞膜Plasma membrane
JsFLA11	245	25.54	6.81	45—180	36	219	22—23	细胞膜Plasma membrane
JsFLA12	425	45.45	5.58	207—343	169, 198	398	30—31	细胞膜Plasma membrane
JsFLA13	272	29.15	5.79	9—111	26	248	24—25	细胞膜Plasma membrane
JsFLA14	223	24.26	8.86	39—133	37	197	—	细胞膜Plasma membrane
JsFLA15	353	39.14	4.80	46—181, 232—321	165	331	24—25	细胞膜Plasma membrane
JsFLA16	255	27.10	9.69	48—183	39, 143	238	25—26	细胞膜Plasma membrane
JsFLA17	159	17.02	5.61	17—120	80	132	—	细胞膜Plasma membrane
JsFLA18	373	40.35	8.30	63—155	55	348	22—23	细胞膜Plasma membrane
JsFLA19	342	38.08	6.21	81—171, 242—331	50, 133	315	—	细胞膜Plasma membrane
JsFLA20	515	56.34	5.76	364—469	144, 345	480	—	细胞膜Plasma membrane
JsFLA21	408	43.36	5.81	227—331	30, 132	383	26—27	细胞膜Plasma membrane
JsFLA22	258	27.63	5.92	53—187	44, 103	230	23—24	细胞膜Plasma membrane
JsFLA23	292	32.29	8.99	115—211	3, 183	255	27—28	细胞膜Plasma membrane
JsFLA24	289	31.24	6.16	115—211	183	263	27—28	细胞膜Plasma membrane

表1

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