棉花FLA基因家族的全基因组鉴定及GhFLA05在棉纤维发育中的功能分析

doi:10.3864/j.issn.0578-1752.2023.23.004

摘要/Abstract

摘要：

【背景】伴随着棉纺织工艺水平的提升和人们对高品质纺织品的追求，提升棉花纤维品质日益重要。类成束阿拉伯半乳糖蛋白（fasciclin-like arabinogalactan proteins，FLAs）在棉纤维起始发育、次生壁合成等过程中可能具有重要作用。【目的】通过对棉花FLA基因家族进行全面鉴定与分析，研究该家族成员的共性特征及特异性表达模式，为FLA在棉纤维发育中的功能研究提供参考。【方法】根据棉花全基因组数据，使用HMMER 3.0对棉花FLA基因家族成员进行鉴定，并通过Pfam、Smart等软件进一步确认。使用ExPASy、TMHMM分析蛋白理化性质及跨膜结构域，应用MEGA、MCScanX、GSDS、MEME、TBtools、Jalview等工具进行进化树构建、染色体定位、共线性分析和蛋白保守结构域序列比对等。通过转录组数据分析陆地棉FLA基因在不同组织中的表达情况。利用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测GhFLAs在不同纤维品质材料的胚珠及不同发育时期纤维中的表达差异。利用病毒诱导的基因沉默（virus induced gene silencing，VIGS）技术验证GhFLA05的功能。【结果】在陆地棉、海岛棉、亚洲棉和雷蒙德氏棉全基因组中分别鉴定出41、40、20和21个FLA家族成员，系统进化树显示，棉花FLA蛋白可以分为4个群组。进一步对陆地棉FLA家族蛋白进行分析，41个成员均具有1—2个AGP-like糖基化区域和1—2个类成束蛋白结构域（fasciclin-like domain，FAS），其中，37个含有信号肽（signal peptide，SP），25个含有糖基化磷脂酰肌醇（glycosylphosphatidy linositol anchored protein，GPI）锚定信号，基因结构和基序组成在各组中相对保守。亚细胞定位显示，GhFLA05_D可能定位在细胞质的内质网，呈聚集状颗粒，GhFLA18_A和GhFLA22在细胞膜/壁、细胞质和细胞核中均有表达。转录组测序结果表明，Group A和Group B中的FLA蛋白主要在纤维中高表达，可能参与了棉纤维发育伸长和次生壁加厚等过程。在纤维品质差异显著的2个材料中，Group A和Group B成员具有相似的表达模式，并主要在纤维次生壁发育阶段、尤其是20—25 DPA时期优势表达；其中，GhFLA05在次生壁增厚期表现出特异性表达，两材料间存在显著差异，在高比强的RIL229的次生壁阶段更早达到最大值，推测GhFLA05可能在调控纤维比强度差异形成中发挥作用。利用VIGS技术沉默GhFLA05后，使棉纤维断裂比强度降低。【结论】在陆地棉、海岛棉、亚洲棉和雷蒙德氏棉中鉴定出122个FLA家族成员，可分为4个群组，不同群组成员间具有较高的结构和功能相似性，并从中鉴定了Group A和Group B成员，可能是主要与棉纤维发育相关的基因。明确家族中GhFLA05是次生壁合成阶段优势表达基因，并与陆地棉不同材料纤维比强度差异形成密切相关。

关键词: 棉花, FLA, 纤维发育, 基因家族, 表达分析

Abstract:

【Background】It is of great importance to improve the quality of cotton fiber to meet the improvement of cotton textile production and the pursue of people for high quality cotton. Fasciclin-like arabinogalactan proteins (FLAs) play an important role in the initial development of cotton fibers and secondary wall synthesis. 【Objective】Comprehensive identification and analysis of cotton FLA gene family members to reveal their common characteristics and specific expression patterns, provided a reference for the function study of FLAs in cotton fiber development.【Method】According to the whole genome data of cotton, members of FLA gene family were identified by HMMER3.0 and further verified by online softwares of Pfam and Smart. Physical and chemical properties and transmembrane domains of these proteins were analyzed by ExPASy and TMHMM. Phylogenetic tree construction, chromosome localization, collinearity analysis and protein conserved domain sequence alignment were conducted and displayed using GSDS, MCScanX, MEGA, MEME, TBtools and Jalview. Expression of FLA genes in different tissues were analyzed by cotton transcriptome data. Expression differences of GhFLAs in different developmental stages of ovules and fibers between different fiber quality materials was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Function of GhFLA05 was verified by virus induced gene silencing (VIGS). 【Result】A total of 41, 40, 20 and 21 FLA family members were identified in G.hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. The phylogenetic tree showed that cotton FLA proteins could be divided into four groups. Gene structure and motif composition were relatively conserved in each group. Further analysis of FLA proteins in Gossypium hirsutum showed that all 41 FLA members had 1-2 AGP-like glycosylation regions and 1-2 fasciclin-like domains (FAS), 37 of which contained signal peptide (SP) and 25 contained glycosylphosphatidylinositol anchored protein (GPI) anchoring signals. Subcellular localization showed that GhFLA05_D showing aggregated granules in the cytoplasm was probably localized in endoplasmic reticulum, and GhFLA18_A and GhFLA22 were expressed in cell membrane/wall, cytoplasm and nucleus. Transcriptome sequencing results showed that FLA proteins in Group A and B were mainly highly expressed in fibers, which may be involved in the process of cotton fiber elongation development and secondary wall thickening. In general, group A and B members had a similar expression pattern in two materials with significant differences in fiber quality and expressed mainly in the secondary wall development stage, especially in 20-25 DPA period. GhFLA05 exhibited specific expression at the secondary wall thickening stage with significant differences between two materials, which expressed with a high maximum value in earlier stage of secondary wall thickening stage in high specific strength material RIL229, suggesting GhFLA05 may take a part in the regulation of cotton fiber strength difference formation. The fiber strength and micronaire value decreased in GhFLA05 gene-silenced cotton plants by VIGS.【Conclusion】A sum of 122 FLA family members were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii, which could be divided into four groups. Members of different groups had high structural and functional similarities, and the genes related to cotton fiber development were identified. It was clarified that GhFLA05 specifically expressed in the secondary wall synthesis stage, and closely related to the difference in fiber strength of different upland cotton materials.

Key words: cotton, FLA, fiber development, gene family, expression analysis

唐丽媛, 蔡肖, 王海涛, 李兴河, 张素君, 刘存敬, 张建宏. 棉花FLA基因家族的全基因组鉴定及GhFLA05在棉纤维发育中的功能分析[J]. 中国农业科学, 2023, 56(23): 4602-4620.

TANG LiYuan, CAI Xiao, WANG HaiTao, LI XingHe, ZHANG SuJun, LIU CunJing, ZHANG JianHong. Genome-Wide Identification of Cotton FLA Gene Family and Functional Analysis of GhFLA05 in Cotton Fiber Development[J]. Scientia Agricultura Sinica, 2023, 56(23): 4602-4620.

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棉种类型 Type of Gossypium	氨基酸数量 No. of amino acid (aa)	分子量 Mw (kDa)	等电点 pI	不稳定系数 Instability index	亲水性平均系数 Grand average of hydropathicity (GRAVY)	跨膜结构域数量No. of predicted TMHs
陆地棉G. hirsutum	241－499	25.61－53.52	5.11－9.27	27.90－54.92	-0.24－0.28	0－3
海岛棉G. barbadense	241－460	25.58－50.83	5.11－9.27	27.90－55.73	-0.24－0.28	0－1
亚洲棉G. arboreum	239－459	25.42－50.71	5.25－9.04	26.95－57.71	-0.25－0.28	0－3
雷蒙德氏棉G. ramondii	239－515	25.50－57.29	5.11－9.41	24.91－54.76	-0.25－0.26	0－2