生姜NHX基因家族成员鉴定及其在硅缓解盐胁迫中的表达特征

doi:10.3864/j.issn.0578-1752.2024.19.011

中国农业科学 ›› 2024, Vol. 57 ›› Issue (19): 3848-3869.doi: 10.3864/j.issn.0578-1752.2024.19.011

生姜NHX基因家族成员鉴定及其在硅缓解盐胁迫中的表达特征

尹军良¹^,²(), 李婧怡¹, 韩硕¹, 杨培华², 马佳伟², 刘奕清², 胡海骏², 朱永兴¹^,²()

¹ 长江大学农学院，湖北荆州 434025
² 长江大学园艺园林学院/香辛园艺植物种质创新与利用湖北省重点实验室/湿地生态与农业利用教育部工程研究中心，湖北荆州 434025

收稿日期:2024-06-09 接受日期:2024-07-22 出版日期:2024-10-01 发布日期:2024-10-09
通信作者:
朱永兴，E-mail：xbnlzyx@163.com
联系方式: 尹军良，E-mail：yinjunliang@yangtzeu.edu.cn。
基金资助:
湖北省重点研发项目(2022BBA0061); 重庆英才优秀科学家项目(CQYC20220101514); 湿地生态与农业利用教育部工程研究中心开放基金(KFT202305); 重庆市自然科学基金项目-面上项目(CSTB2022NSCQ-MSX1588)

Identification of Ginger (Zingiber officinale Roscoe) NHX Gene Family Members and Characterization of Their Expression Patterns in Silicon Alleviating Salt Stress

YIN JunLiang¹^,²(), LI JingYi¹, HAN Shuo¹, YANG PeiHua², MA JiaWei², LIU YiQing², HU HaiJun², ZHU YongXing¹^,²()

¹ College of Agriculture, Yangtze University, Jingzhou 434025, Hubei
² College of Horticulture and Gardening, Yangtze University/ Hubei Key Laboratory of Spices & Horticultural Plant Germplasm Innovation & Utilization/Engineering Research Center of Wetland Ecology and Agricultural Use, Ministry of Education, Jingzhou 434025, Hubei

Received:2024-06-09 Accepted:2024-07-22 Published:2024-10-01 Online:2024-10-09

摘要/Abstract

摘要：

【目的】系统分析生姜NHX基因家族成员的基本特征，探究其在生姜不同组织、处理下的表达模式，为进一步研究ZoNHXs功能提供理论基础。【方法】以拟南芥NHX蛋白序列为参考序列，与生姜基因组进行BLAST比对，筛选出包含Na⁺/H⁺ exchange保守结构域的蛋白序列，得到生姜ZoNHX家族成员。使用MEGA 7.0构建拟南芥和生姜NHX家族成员的系统发育树，利用ExPASy ProtParam、GSDS、MEME、PHYRE2、SOPMA和TBtools对ZoNHXs的理化性质、基因结构、保守基序、二级结构和三级结构、染色体定位、基因共线性及表达模式进行分析。借助RT-qPCR分析ZoNHXs在对照（CK）、盐胁迫（NaCl）、盐胁迫+纳米硅（NaCl+SiNP）处理的生姜根系和叶片中的表达特征，并测定生姜各个组织内的Na⁺、K⁺离子含量。【结果】从生姜基因组中共鉴定出15个ZoNHX基因家族成员，根据其染色体位置分别命名为ZoNHX1—ZoNHX15。根据系统发育关系和亚细胞定位分析，将ZoNHXs归为液泡膜（vacuole，Vac）、核内体膜（endosome，Endo）和质膜（plasma membrane，PM）3个亚组。蛋白质特征分析结果表明，ZoNHXs的相对分子量介于26.01—163.59 kDa，氨基酸序列长度介于231—1 459 aa。亚细胞定位分析发现，11个ZoNHXs定位在液泡，ZoNHX14定位于细胞膜和细胞核，ZoNHX1、ZoNHX7和ZoNHX9分别定位于细胞质、细胞膜和叶绿体。信号肽预测发现，只有ZoNHX1为含有信号肽的分泌蛋白，其余均为非分泌蛋白。顺式作用元件分析显示，ZoNHXs启动子区域包含生长发育响应元件、激素响应元件和胁迫响应元件。转录组数据分析表明，15个ZoNHXs在生姜叶片和根系中均有表达；其中，ZoNHX12在生姜各组织和各非生物胁迫下表达水平相对较高，ZoNHX14在生姜各生长时期及低温下表达量显著上调。RT-qPCR结果显示，与CK相比，除ZoNHX8、ZoNHX11外，其余基因在盐胁迫后的根系和叶片中表达量均显著上调。同单独盐胁迫相比，NaCl+SiNP100处理后，除根系和叶片ZoNHX8、ZoNHX11的表达水平显著上调外，其余基因的表达水平均下调，表明ZoNHXs参与生姜对盐胁迫的响应过程，且ZoNHXs的表达受外源SiNP的调控。Na⁺、K⁺离子含量、K⁺/Na⁺比值及运输选择系数S_K⁺_/Na⁺结果表明，盐胁迫处理下生姜根茎、茎、叶中的Na⁺含量较CK上升，K⁺/Na⁺比值及运输选择系数下降，NaCl+SiNP100处理的根、根茎、茎、叶中的Na⁺含量显著下降，K⁺/Na⁺比值及运输选择系数上升。表明ZoNHXs可能参与生姜各组织的Na⁺、K⁺转运调控，缓解盐胁迫损伤。【结论】生姜基因组中15个ZoNHXs可分为3个亚组，分布在6条染色体上；ZoNHX1—ZoNHX15的表达均受盐胁迫诱导；SiNP100预处理可调控ZoNHXs的表达，降低植株体内Na⁺含量，提高生姜根、根茎、茎和叶片中的K⁺/Na⁺比值和K⁺、Na⁺运输选择性系数，从而缓解植株盐胁迫。

关键词: 生姜, NHX基因家族, 盐胁迫, 表达分析, 转录组

Abstract:

【Objective】 Systematic analysis of the basic features of the ZoNHX family members in ginger and exploration of their expression patterns in different tissues and treatments of ginger can provide a theoretical foundation for further research on the function of ZoNHXs. 【Method】The Arabidopsis thaliana NHX protein sequences were used as reference sequences to compare with the ginger genome to obtain the ginger ZoNHXs. The phylogenetic tree containing Arabidopsis and ginger NHX family members were constructed based on the neighbor-joining method by MEGA 7.0. Physicochemical properties, gene structure, conserved motifs, secondary structure and tertiary structure of ZoNHXs were analyzed by using ExPASy ProtParam software, GSDS, MEME online website, PHYRE 2 and SOPMA, while chromosomal localization, intragenomic covariance and expression patterns of ZoNHXs were analyzed by using TBtools software. The gene expression levels of ZoNHXs in different treatments including CK, NaCl, and NaCl+SiNP100, and different ginger tissues were analyzed using RT-qPCR. The Na⁺ and K⁺ contents of ginger tissues was determined. 【Result】A total of 15 NHX gene family members were identified from the ginger genome, and were named as ZoNHX1-ZoNHX15 based on their chromosomal locations. The ZoNHXs were classified into three subgroups according to phylogenetic relationship and subcellular localization, including vesicular membranes (Vac, vacuole), endosomal membranes in the nucleus (Endo, endosome) and plasma membranes (PM, plasma). Protein characterization analysis showed that the relative molecular weights of ZoNHXs ranged from 26.01 kDa to 163.59 kDa, and the protein lengths ranged from 231 to 1 459 aa. The results of subcellular localization prediction showed that 11 ZoNHXs were distributed on the vacuole, ZoNHX14 were distributed both on the cell membrane and nucleus, ZoNHX1, ZoNHX7 and ZoNHX9 were respectively distributed on cytoplasm, cell membrane, and chloroplast. Signal peptide prediction showed that only ZoNHX1 contained a signal peptide and was a secreted protein, while other ZoNHX family members were non-secretory proteins. Cis-acting element analysis showed that the promoter regions of the ZoNHXs contain growth and development response elements, hormone response elements, and stress response elements. Transcriptome analysis showed that all ZoNHXs were detected to be expressed in ginger leaf and root, among which, ZoNHX12 was highly expressed in all tissues of ginger and under different abiotic stresses, and ZoNHX14 was significantly up-regulated in all growth periods of ginger and at low temperatures. RT-qPCR results showed that, compared with the CK, all genes were significantly up-regulated in roots and leaves after salt stress except for ZoNHX8 and ZoNHX11. Compared with the NaCl treatment alone, NaCl+SiNP100 decreased the expression levels of all genes except for ZoNHX8 and ZoNHX11, the expression levels of which were significantly up-regulated. These indicated that ZoNHXs were involved in the response process of ginger to salt stress and the expression of ZoNHXs were also regulated by exogenous SiNP100. The results of Na⁺, K⁺ion content, K⁺/Na⁺ratio and transport selection coefficient S_K⁺_/Na⁺ showed that the Na⁺ content in ginger rhizomes, stems, and leaves under salt treatment was increased compared with CK, and the K⁺/Na⁺ ratio and transport selection coefficient were decreased. NaCl+SiNP100 treatment significantly decreased Na⁺content in roots, rhizomes, stems and leaves, whereases increased K⁺/Na⁺ ratio and transport selection coefficient. 【Conclusion】 The ginger genome contains 15 ZoNHXs and divides into three subgroups. RT-qPCR results showed that the expression of ZoNHX1-ZoNHX15 were all induced by salt stress. SiNP100 pretreatment could alleviate plant salt stress through reducing the Na⁺ content and increase the K⁺/Na⁺ ratio and K⁺ and Na⁺ content of the ginger roots, rhizomes, stems, and leaves by regulating the expression of ZoNHXs.

Key words: ginger, NHX gene family, salt stress, expression analysis, transcriptome

尹军良, 李婧怡, 韩硕, 杨培华, 马佳伟, 刘奕清, 胡海骏, 朱永兴. 生姜NHX基因家族成员鉴定及其在硅缓解盐胁迫中的表达特征[J]. 中国农业科学, 2024, 57(19): 3848-3869.

YIN JunLiang, LI JingYi, HAN Shuo, YANG PeiHua, MA JiaWei, LIU YiQing, HU HaiJun, ZHU YongXing. Identification of Ginger (Zingiber officinale Roscoe) NHX Gene Family Members and Characterization of Their Expression Patterns in Silicon Alleviating Salt Stress[J]. Scientia Agricultura Sinica, 2024, 57(19): 3848-3869.

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基因ID Gene ID	名称 Name	引物序列Primer sequence (5′-3′)		长度 Length (bp)
基因ID Gene ID	名称 Name	正向Forword	反向Reverse	长度 Length (bp)
Maker00056825	ZoNHX1	AATGCCTGGTTTGCTTCCTC	GCTTGTTGGGCTTGCTGTG	99
Maker00009627	ZoNHX2	AGATGGGAGGGTTCACGAG	GACAATGTACCCCCGATCC	116
Maker00078687	ZoNHX3	CAAACCCTTCCGCTTCTCC	GACGACGAGGCTCCGACTA	133
Maker00077763	ZoNHX4	TGCCCTGGCATTTAGTCAG	AGGTCGTCCGACTTCCATC	166
Maker00035912	ZoNHX5	GACAAGAGGTTACAGAGGGATATG	CACCGAAAGTGTAGAAGGAAAG	151
Maker00016804	ZoNHX6	TTGACTCGTCGGCTTTGATC	CTGACCCATGACTATTTGGTAGAA	131
Maker00016508	ZoNHX7	CGATGGTGGTTCACAGAGG	ACCGACGCTGAGCTACCTT	155
Maker00016634	ZoNHX8	ATCCTGTTGCTGTCGTTGC	ATTGCAGTCCCATCGTTCA	143
Maker00016696	ZoNHX9	AGCATGTCGGTTTGCTCAG	TTCTGCAATGGCTTCTTGG	162
Maker00029358	ZoNHX10	TTGTGCATCCCCACTGTTT	AATAGGGGGCAGCAAGAAC	177
Maker00028403	ZoNHX11	CTGGTCGCATGTTGGATTC	TGAAGGGCAAGTGCAAAAG	134
Maker00058140	ZoNHX12	GCTGGGGATCTCTCAGCTC	CAGGTGGCCAATAACGATG	154
Maker00022420	ZoNHX13	TGGAATGGATGCATTGGAC	CAAGCCATGTCCATTTTCG	119
Maker00061543	ZoNHX14	TTTCTCCCGACCATTCTCC	GACGTCGTCAACACCAACC	170
Maker00007292	ZoNHX15	GCTGCGTCTTCCACTCAAC	CCCTCCCCGAAGACAATAC	149
MTCONS_00009943	RBP	CCTATGAAGCGTAGAAACACAAG	GGAAGGACAACATCCCAAATC	123

基因名称 Gene name	基因ID Gene ID	蛋白长度 Protein length (aa)	分子量 Molecular weight (kDa)	等电点 pI	亲水指数 Gravy	不稳定系数 Instability index	亚细胞预测 Predicted location	信号肽 Signal
ZoNHX1	Maker00056825	231	26.01	5.96	0.238	39.92	细胞质 Cytoplasm	有 Yes
ZoNHX2	Maker00009627	1459	163.59	8.88	-0.036	42.16	液泡 Vacuole	无 No
ZoNHX3	Maker00078687	315	34.76	7.08	0.388	46.86	液泡 Vacuole	无 No
ZoNHX4	Maker00077763	425	47.62	8.96	0.394	44.93	液泡 Vacuole	无 No
ZoNHX5	Maker00035912	539	58.62	5.43	0.306	44.86	液泡 Vacuole	无 No
ZoNHX6	Maker00016804	543	60.23	8.99	-0.049	43.39	液泡 Vacuole	无 No
ZoNHX7	Maker00016508	990	111.68	9.08	-0.055	47.28	细胞膜 Cell membrane	无 No
ZoNHX8	Maker00016634	318	34.14	5.42	0.745	33.06	液泡 Vacuole	无 No
ZoNHX9	Maker00016696	833	93.58	6.13	-0.137	41.12	叶绿体 Chloroplast	无无 No
ZoNHX10	Maker00029358	257	28.72	5.27	0.854	46.73	液泡 Vacuole	无 No
ZoNHX11	Maker00028403	292	32.51	6.66	0.273	42.53	液泡 Vacuole	无 No
ZoNHX12	Maker00058140	617	68.4	6.64	0.521	36.21	液泡 Vacuole	无 No
ZoNHX13	Maker00022420	324	36.19	9.24	-0.008	53.85	细胞膜、细胞核 Cell membrane, Nucleus	无 No
ZoNHX14	Maker00061543	683	75.61	9.47	0.443	44.91	液泡 Vacuole	无 No
ZoNHX15	Maker00007292	450	49.88	8.48	0.420	34.1	液泡 Vacuole	无 No

蛋白Protein	α-螺旋 Alpha helix (%)	延伸链 Extended strand (%)	β-转角 Beta turn (%)	不规则卷曲 Random coil (%)
ZoNHX3	39.05	21.59	10.48	28.89
ZoNHX1	44.16	19.91	6.93	29.00
ZoNHX9	45.74	12.48	5.28	36.49
ZoNHX7	45.96	16.77	9.70	27.58
ZoNHX6	41.25	18.23	9.02	31.49
ZoNHX8	54.09	19.81	6.29	19.81
ZoNHX10	42.80	20.23	3.50	33.46
ZoNHX11	48.63	11.30	4.45	35.62
ZoNHX5	44.34	16.88	3.15	35.62
ZoNHX13	40.12	19.75	9.26	30.86
ZoNHX15	41.56	18.67	4.22	35.56
ZoNHX4	46.12	19.29	3.53	31.06
ZoNHX14	40.56	18.74	4.69	36.02
ZoNHX12	41.98	22.37	5.53	30.31
ZoNHX2	34.82	23.92	11.38	29.88

处理 Treatment	根Root	根茎Tuber	茎Stem	叶Leaf
CK	278.93±27.39a	64.59±2.77a	2779.50±17.65a	4040.83±1206.794a
NaCl	7.00±0.67b	4.87±0.21c	17.65±1.25b	16.84±3.20b
SiNP100+NaCl	14.10±0.43b	12.18±1.35b	46.74±4.75b	105.96±7.55b

处理 Treatment	根→根茎(S_K⁺_/Na⁺) Root→Tuber	根茎→茎(S_K⁺_/Na⁺) Tuber→Stem	茎→叶(S_K⁺_/Na⁺) Stem→Leaf
CK	0.23±0.02c	43.16±7.10a	1.44±0.33b
NaCl	0.70±0.06b	3.63±0.37b	0.97±0.24b
SiNP100+NaCl	0.86±0.08a	3.87±0.60b	2.28±0.19a

生姜NHX基因家族成员鉴定及其在硅缓解盐胁迫中的表达特征

Identification of Ginger (Zingiber officinale Roscoe) NHX Gene Family Members and Characterization of Their Expression Patterns in Silicon Alleviating Salt Stress

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