铁皮石斛DoNPF基因家族的挖掘、鉴定及表达分析

doi:10.16178/j.issn.0528-9017.20240611

摘要/Abstract

摘要：

低温是限制植物生产力和地理分布的重要环境因子之一,严重影响植物的生长和发育。挖掘抗寒种质,深入研究其作用机理,对提高作物的抗寒性十分重要。铁皮石斛是我国传统名贵中药材,具有生津养胃、滋阴清热、润肺益肾等独特功效,被列为“中华九大仙草”之首。随着仿野生种植技术的发展和铁皮石斛种植区域的逐渐北移,选育铁皮石斛抗寒品种尤为重要。本研究通过深入挖掘铁皮石斛低温处理的转录组数据,获得参与低温响应的关键DoNPF基因。基于已发表的铁皮石斛基因组数据及转录组数据,利用生物信息学方法对DoNPF基因家族成员进行鉴定,并对其基因结构、基因表达进行分析,筛选出8个参与低温响应的显著(p<0.05)差异表达的DoNPF基因,对提高铁皮石斛的抗寒性以及培育铁皮石斛抗寒品种具有十分重要的意义。

关键词: 铁皮石斛, DoNPF基因家族, 低温, 全基因组鉴定

Abstract:

Low temperature is one of the crucial environmental factors that limit plant productivity and geographical distribution, severely affecting plant growth and development. Mining cold-resistant germplasm and conducting in-depth research on its mechanism of action are of great significance for improving the cold resistance of crops. Dendrobium officinale is a traditional and precious Chinese medicinal herb in China, with unique effects such as promoting saliva secretion and nourishing the stomach, nourishing yin and clearing heat, and moistening the lungs and tonifying the kidneys. It is ranked first among the “nine great immortal herbs of China”. With the development of wild-simulation cultivation technology and the gradual northward expansion of Dendrobium officinale planting areas, the breeding of cold-resistant Dendrobium officinale varieties is particularly important. In this study, transcriptome data of Dendrobium officinale under low-temperature treatment were deeply mined to obtain key DoNPF genes involved in low-temperature response. Based on the published genome data and transcriptome data of Dendrobium officinale, bioinformatics methods were used to identify members of the DoNPF gene family, and their gene structures and gene expressions were analyzed. Finally, 8 DoNPF genes with significantly (p<0.05) differential expression involved in low-temperature response were screened out. This study is of great significance for improving the cold resistance of Dendrobium officinale and breeding cold-resistant Dendrobium officinale varieties.

Key words: Dendrobium officinale, DoNPF gene family, low temperature, genome-wide identification

中图分类号:

S567

付曼曼, 郭方其, 吴超. 铁皮石斛DoNPF基因家族的挖掘、鉴定及表达分析[J]. 浙江农业科学, 2025, 66(12): 2944-2951.

FU Manman, GUO Fangqi, WU Chao. Mining, identification, and expression analysis of the DoNPF gene family in Dendrobium officinale[J]. Journal of Zhejiang Agricultural Sciences, 2025, 66(12): 2944-2951.

图/表 6

图1 铁皮石斛中与低温相关的差异表达基因 (a)火山图显示了与常温条件相比,在低温条件下基因的差异表达,红色代表上调基因,蓝色代表下调基因。(b)热图显示了每个样本中差异表达基因的表达量(对每个基因在所有样本中的表达量进行Z-score标准化,以突出每个基因在不同条件下相对表达水平),对不同样本和不同基因进行了聚类分析。

Fig.1 Differential expression genes related to low temperature in Dendrobium officinale

图2 铁皮石斛中差异表达基因(DEG)的蛋白质相互作用(PPI)网络 (a)DEG对应水稻基因的PPI,红色表示Cytoscape软件预测的DEG基因簇。(b)红色标注的DEG基因簇的蛋白质相互作用。

Fig.2 Protein-protein interactions of differential expression genes (DEG) of Dendrobium officinale

图3 NPF基因家族编码蛋白的系统发育树系统发育树由拟南芥、水稻和铁皮石斛的NPF基因家族编码的蛋白序列构建。硝酸盐转运蛋白 (NRT)基因家族编码的蛋白序列被认为是外类群,NPF基因家族编码的蛋白序列的不同亚群用不同颜色表示。

Fig.3 Phylogenetic tree of NPF gene family encoding proteins

图4 DoNPF基因结构

Fig.4 Gene structure of DoNPF

图5 DoNPF基因在常温和低温处理下的表达情况 “**”表示不同温度下基因表达差异极显著(p<0.01)。图6同。

Fig.5 Expression of DoNPF under normal and low-temperature treatment

图6 低温处理下显著差异表达的DoNPF基因

Fig.6 DoNPF genes significantly differentially expressed under low-temperature treatment

参考文献 23

[1]	YAN L, WANG X, LIU H, et al. The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb[J]. Molecular Plant, 2015, 8(6): 922-934.
[2]	邹晖, 林江波, 李海明, 等. 铁皮石斛人工栽培技术[J]. 福建农业, 2016(12): 38-39.
[3]	蒋新建, 陈玉哲. 郑州地区铁皮石斛人工种植管理技术[J]. 河南农业, 2018(30): 48-49.
[4]	DANIEL H. Molecular and integrative physiology of intestinal peptide transport[J]. Annual Review of Physiology, 2004, 66: 361-384.
[5]	NEWSTEAD S, DREW D, CAMERON A D, et al. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2[J]. EMBO Journal, 2011, 30(2): 417-426.
[6]	FEI Y J, KANAI Y, NUSSBERGER S, et al. Expression cloning of a mammalian proton-coupled oligopeptide transporter[J]. Nature, 1994, 368(6471): 563-566.
[7]	TSAY Y F, SCHROEDER J I, FELDMANN K A, et al. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter[J]. Cell, 1993, 72(5): 705-713.
[8]	LÉRAN S, VARALA K, BOYER J C, et al. A unified nomenclature of nitrate transporter 1/peptide transporter family members in plants[J]. Trends in Plant Science, 2014, 19(1): 5-9.
[9]	PARKER J L, NEWSTEAD S. Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1[J]. Nature, 2014, 507(7490): 68-72.
[10]	WEN J, LI P F, RAN F, et al. Genome-wide characterization, expression analyses, and functional prediction of the NPF family in Brassica napus[J]. BMC Genomics, 2020, 21(1): 871.
[11]	LI J, LIU H L, XIA W W, et al. De novo transcriptome sequencing and the hypothetical cold response mode of Saussurea involucrata in extreme cold environments[J]. International Journal of Molecular Sciences, 2017, 18(6): 1155.
[12]	NOUR-ELDIN H H, ANDERSEN T G, BUROW M, et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds[J]. Nature, 2012, 488(7412): 531-534.
[13]	ZHAO X B, HUANG J Y, YU H H, et al. Genomic survey, characterization and expression profile analysis of the peptide transporter family in rice (Oryza sativa L.)[J]. BMC Plant Biology, 2010, 10: 92.
[14]	YANG X H, XIA X Z, ZENG Y, et al. Genome-wide identification of the peptide transporter family in rice and analysis of the PTR expression modulation in two near-isogenic lines with different nitrogen use efficiency[J]. BMC Plant Biology, 2020, 20(1): 193.
[15]	BUCHNER P, HAWKESFORD M J. Complex phylogeny and gene expression patterns of members of the nitrate transporter 1/peptide transporter family (npf) in wheat[J]. Journal of Experimental Botany, 2014, 65(19): 5697-5710.
[16]	WANG Q, LIU C H, DONG Q L, et al. Genome-wide identification and analysis of apple nitrate transporter 1/peptide transporter family (npf) genes reveals MdNPF6.5 confers high capacity for nitrogen uptake under low-nitrogen conditions[J]. International Journal of Molecular Sciences, 2018, 19(9): 2761.
[17]	LI H, YU M, DU X Q, et al. NRT1.5/NPF7.3 functions as a proton-coupled H⁺/K⁺ antiporter for K⁺ loading into the xylem in Arabidopsis[J]. The Plant Cell, 2017, 29(8): 2016-2026.
[18]	WATANABE S, TAKAHASHI N, KANNO Y, et al. The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism[J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(49): 31500-31509.
[19]	TONG W, IMAI A, TABATA R, et al. Polyamine resistance is increased by mutations in a nitrate transporter gene NRT1.3 (AtNPF6.4) in Arabidopsis thaliana[J]. Frontiers in Plant Science, 2016, 7: 834.
[20]	HE Y N, XI X J, ZHA Q, et al. Ectopic expression of a grape nitrate transporter VvNPF6.5 improves nitrate content and nitrogen use efficiency in Arabidopsis[J]. BMC Plant Biology, 2020, 20(1): 549.
[21]	CHEN H Y, LIN S H, CHENG L H, et al. Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner[J]. The Plant Cell, 2021, 33(5): 1492-1505.
[22]	LI B, QIU J E, JAYAKANNAN M, et al. AtNPF2.5 modulates chloride (Cl^-) efflux from roots of Arabidopsis thaliana[J]. Frontiers in Plant Science, 2017, 7: 2013.
[23]	XIA X D, FAN X R, WEI J, et al. Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport[J]. Journal of Experimental Botany, 2015, 66(1): 317-331.