苹果NCL家族基因与根系细胞钙离子浓度变化的关系

doi:10.16420/j.issn.0513-353x.2021-1267

摘要/Abstract

摘要：

基于拟南芥类Na⁺/Ca²⁺交换蛋白（NCX-Like，NCL）序列以及苹果、葡萄、草莓、桃和梨全基因组信息，分析了NCL家族特征、苹果MdNCL表达以及不同处理下湖北海棠根系MhNCL的表达水平与细胞Ca²⁺浓度变化的关系。结果表明，NCL家族成员在进化中高度保守，苹果基因组中的4个MdNCL的启动子上均含有胁迫响应及激素应答相关的顺式作用元件，且在苹果各器官均有不同程度表达。依据MdNCL保守结构域，从湖北海棠中确定4个相应的MhNCL基因（MhNCL-1、MhNCL-2、MhNCL-3和 MhNCL-4），其中MhNCL-1、MhNCL-3和MhNCL-4的表达水平在NaCl和CaCl₂处理下基本呈现“变化不大—上升—下降”的规律，在ABA和IAA处理下表现出“变化不大—上升—下降—上升”的特征。根系细胞Ca²⁺浓度在NaCl和CaCl₂处理下呈现“升高—降低—小幅度升高”的规律，在ABA和IAA处理下呈现“升高—降低—升高—降低”的趋势，初步表明根系MhNCL的表达水平与根系Ca²⁺浓度变化存在一定相关性。

关键词: 苹果, NCL基因, 全基因组鉴定, Ca²⁺浓度

Abstract:

Na⁺/Ca²⁺exchange-like proteins（NCX-Like，NCL）take part in the transmembrane transport of Ca²⁺ and other cations in Arabidopsis. The traits of NCL family gene and expression patterns of apple MdNCL based on Arabidopsis NCL sequences and genome-wide information of apple，grape，strawberry，peach and pear were analyzed；and the relationship between MhNCL expression and cellular Ca²⁺ concentration in the roots of Malus hupehensis Rhed. under different treatments were investigated. Results showed that the NCL family members were highly conserved in evolution；four MdNCLs originated from apple genome all contained cis-acting components of abiotic stress response and hormone response on their promoters and had organ-specific expression patterns. Based on the conserved structural domains of MdNCL，four MhNCLs（MhNCL-1，MhNCL-2，MhNCL-3 and MhNCL-4）were identified in M. hupehensis. Expression levels of MhNCL-1，MhNCL-3 and MhNCL-4 showed a“little change-increase-decrease-increase”pattern under NaCl and CaCl₂treatments，and showed a“little change-increase-decrease-increase”pattern under ABA and IAA treatments. The Ca²⁺concentration in root cells showed an“increase-decrease-small increase”trend under NaCl and CaCl₂treatments，and an“increase-decrease-increase-decrease”pattern under ABA and IAA treatments，which indicated that there was a correlation between the expression levels of MhNCLs and changes in cellular Ca²⁺concentration in root.

Key words: apple, NCL gene, genome-wide identification, Ca²⁺concentration

中图分类号:

S661.1

宁源生, 李欢, 宋建飞, 于婷婷, 韩梦圆, 彭璐琳, 贾竣淇, 张玮玮, 杨洪强. 苹果NCL家族基因与根系细胞钙离子浓度变化的关系[J]. 园艺学报, 2023, 50(3): 475-484.

NING Yuansheng, LI Huan, SONG Jianfei, YU Tingting, HAN Mengyuan, PENG Lulin, JIA Junqi, ZHANG Weiwei, YANG Hongqiang. Characterization of NCL Family Genes in Malus and Their Relationship with Cellular Calcium Concentration in Root[J]. Acta Horticulturae Sinica, 2023, 50(3): 475-484.

图/表 7

参考文献 28

[1]	Batistič O, Kudla J. 2012. Analysis of calcium signaling pathways in plants. Biochim Biophys Acta, 1820 (8):1283-1293. doi: 10.1016/j.bbagen.2011.10.012 pmid: 22061997
[2]	Blaustein M P, Lederer W J. 1999. Sodium/calcium exchange:its physiological implications. Physiological Reviews, 793:763-854.
[3]	Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. TBtools:an integrative toolkit developed for interactive analyses of big biological data. Mol Plant, 13 (8):1194-1202. doi: 10.1016/j.molp.2020.06.009 URL
[4]	Dipolo R, Beauge L. 2006. Sodium/calcium exchanger:influence of metabolic regulation on ion carrier interactions. Physiol Rev, 86 (1):155-203. doi: 10.1152/physrev.00018.2005 URL
[5]	Do T D, Chen H, Hien V T, Hamwieh A, Yamada T, Sato T, Yan Y, Cong H, Shono M, Suenaga K, Xu D. 2016. NCL synchronously regulates Na⁺,K⁺,and Cl^- in soybean and greatly increases the grain yield in saline field conditions. Sci Rep, 6:19147. doi: 10.1038/srep19147
[6]	Dodd A N, Kudla J, Sanders D. 2010. The language of calcium signaling. Annu Rev Plant Biol, 61:593-620. doi: 10.1146/annurev-arplant-070109-104628 pmid: 20192754
[7]	Edel K H, Marchadier E, Brownlee C, Kudla J, Hetherington A M. 2017. The evolution of calcium-based signalling in plants. Curr Biol, 27 (13):R667-R679. doi: 10.1016/j.cub.2017.05.020 URL
[8]	Emery L, Whelan S, Hirschi K D, Pittman J K. 2012. Protein phylogenetic analysis of Ca²⁺/cation antiporters and insights into their evolution in plants. Front Plant Sci, 3:1.
[9]	Hu B, Jin J P, Guo A Y, Zhang H, Luo J C, Gao G. 2015. GSDS 2.0:an upgraded gene feature visualization server. Bioinformatics, 31 (8):1296-1297. doi: 10.1093/bioinformatics/btu817 URL
[10]	Jin H Q, Xing M G, Cai C M, Li S. 2019. B-box proteins in arachis duranensis:genome-wide characterization and expression profiles analysis. Agronomy, 10 (1):1-17. doi: 10.3390/agronomy10010001 URL
[11]	Khananshvili D. 2020. Basic and editing mechanisms underlying ion transport and regulation in NCX variants. Cell Calcium, 85:102131. doi: 10.1016/j.ceca.2019.102131 pmid: 31794905
[12]	Kumar S, Stecher G, Tamura K. 2016. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 33 (7):1870-1874. doi: 10.1093/molbev/msw054 URL
[13]	Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. 2002. PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 30 (1):325-327.
[14]	Letunic I, Bork P. 2018. 20 years of the SMART protein domain annotation resource. Nucleic Acids Re, 46 (D1):D493-D496.
[15]	Li P H, Zhang G Y, Gonzales N, Guo Y Q, Hu H H, Park S, Zhao J. 2016. Ca²⁺-regulated and diurnal rhythm-regulated Na⁺/Ca²⁺ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis. Plant Cell Environ, 39 (2):377-392. doi: 10.1111/pce.v39.2 URL
[16]	Liu Y, Wang L, Xing X, Sun L P, Pan J W, Kong X P, Zhang M Y, Li D Q. 2013. ZmLEA3,a multifunctional group 3 LEA protein from maize(Zea mays L.),is involved in biotic and abiotic stresses. Plant and Cell Physiology, 54 (6):944-959. doi: 10.1093/pcp/pct047 URL
[17]	Mao K, Yang J, Wang M, Liu H Y, Guo X, Zhao S, Dong Q L, Ma F W. 2021. Genome-wide analysis of the apple CaCA superfamily reveals that MdCAX proteins are involved in the abiotic stress response as calcium transporters. BMC Plant Biology, 21 (1):81. doi: 10.1186/s12870-021-02866-1 pmid: 33557757
[18]	Marchler-Bauer A, Bo Y, Han L Y, He J, Lanczycki C J, Lu S N, Chitsaz F, Derbyshire M K, Geer R C, Gonzales N R, Gwadz M, Hurwitz D I, Lu F, Marchler G H, Song J S, Thanki N, Wang Z X, Yamashita R A, Zhang D C, Zheng C J, Geer L Y, Bryant S H. 2017. CDD/SPARCLE:functional classification of proteins via subfamily domain architectures. Nucleic Acids Res, 45 (D1):D200-D203 doi: 10.1093/nar/gkw1129 URL
[19]	Meng D, Li Y Y, Bai Y, Li M J, Cheng L L. 2016. Genome-wide identification and characterization of wrky transcriptional factor family in apple and analysis of their responses to waterlogging and drought stress. Plant Physiology and Biochemistry, 103:71-83. doi: 10.1016/j.plaphy.2016.02.006 pmid: 26970718
[20]	Pittman J K, Hirschi K D. 2016. CAX-ing a wide net:cation/H⁺ transporters in metal remediation and abiotic stress signalling. Plant Biology, 18 (5):741-749. doi: 10.1111/plb.12460 pmid: 27061644
[21]	Qiu L, Hu S, Wang Y, Qu H. 2021. Accumulation of abnormal amyloplasts in pulp cells induces bitter pit in Malus domestica. Front Plant Sci, 23 (12):738726.
[22]	Singh A K, Kumar R, Tripathi A K, Gupta B K, Pareek A, Singla-Pareek S L. 2015. Genome-wide investigation and expression analysis of sodium/calcium exchanger gene family in rice and Arabidopsis. Rice, 8 (1):54. doi: 10.1186/s12284-015-0054-5 pmid: 26134707
[23]	Song J F, Yang F, Xun M, Xu L X, Yang H Q. 2020. Genome-wide identification and characterization of vacuolar processing enzyme gene family and diverse expression under stress in apple(Malus × domestica). Front Plant Sci, 26;11:626.
[24]	Spalding E P, Harper J F. 2011. The ins and outs of cellular Ca²⁺transport. Curr Opin Plant Biol, 14 (6):715-720. doi: 10.1016/j.pbi.2011.08.001 pmid: 21865080
[25]	Su Y L, He H H, Wang P, Ma Z H, Mao J, Chen B H. 2021. Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid Aux/IAA gene family in apple Malus × domestica. Gene, 768:145302. doi: 10.1016/j.gene.2020.145302 URL
[26]	Tian Y, Dong Q L, Ji Z R, Chi F M, Cong P H, Zhou Z S. 2015. Genome-wide identification and analysis of the MADS-box gene family in apple. Gene, 555:277-290. doi: 10.1016/j.gene.2014.11.018 pmid: 25447908
[27]	Wang P, Li Z W, Wei J S, Zhao Z L, Sun D Y, Cui S. 2012. A Na⁺/Ca²⁺exchanger-like protein AtNCL involved in salt stress in Arabidopsis. Journal of Biological Chemistry, 287 (53):44062-44070. doi: 10.1074/jbc.M112.351643 URL
[28]	Wu Q, Huang L P, Su N N, Shabala L, Wang H Y, Huang X, Wen R Y, Yu M, Cui J, Shabala S. 2020. Calcium-dependent hydrogen peroxide mediates hydrogen-rich water-reduced cadmium uptake in plant roots. Plant Physiol, 183 (3):1331-1344. doi: 10.1104/pp.20.00377 pmid: 32366640

基因名称 Gene name	正向引物（5′-3′） Forward primer sequence	反向引物（5′-3′） Revers primer sequence
MhNCL-1	GGTGAACCTGATGCAGACAT	TCAGGCTTACTGATACGGGA
MhNCL-2	GTCTAACAACTCCACTGTGC	GCAATAACTCGCTCCCATTC
MhNCL-3	AGCTTCTCAACAGCTACGAG	ACCAGACCCAAGAATACTGC
MhNCL-4	CAAGTTTGGTAAGATTCCGGG	CTGTTTCGTCCCATGTCCTT
MdActin	ATTCAAGTATGCCTGGGTGC	CAGTCAGCCTGTGATGTTCC

基因名称 Gene name	正向引物（5′-3′） Forward primer sequence	反向引物（5′-3′） Revers primer sequence
MhNCL-1	GGTGAACCTGATGCAGACAT	TCAGGCTTACTGATACGGGA
MhNCL-2	GTCTAACAACTCCACTGTGC	GCAATAACTCGCTCCCATTC
MhNCL-3	AGCTTCTCAACAGCTACGAG	ACCAGACCCAAGAATACTGC
MhNCL-4	CAAGTTTGGTAAGATTCCGGG	CTGTTTCGTCCCATGTCCTT
MdActin	ATTCAAGTATGCCTGGGTGC	CAGTCAGCCTGTGATGTTCC

物种 Species	基因名称 Gene name	基因登录号 Gene ID	氨基酸 aa	等电点 pI	分子量/kD Molecular weight	染色体 Chromosome	亚细胞定位 Subcellular localization
苹果	MdNCL-1	MD03G1238600	586	5.39	63.75	Chr3	细胞膜 Cell membrane
Malus × domestica	MdNCL-2	MD03G1238700	600	5.30	66.03	Chr3	细胞膜 Cell membrane
	MdNCL-3	MD11G1258400	584	5.01	63.26	Chr11	细胞膜 Cell membrane
	MdNCL-4	MD17G1233800	585	5.34	64.56	Chr17	细胞膜 Cell membrane
拟南芥 Arabidopsis thaliana	AtNCL	AT1G53210.1	585	5.15	63.42	Chr1	细胞膜 Cell membrane
葡萄	VvNCL-1	VIT_212s0028g02570	577	5.73	62.98	Chr12	细胞膜 Cell membrane
Vitis vinifera	VvNCL-2	VIT_219s0014g01740	579	5.21	63.10	Chr19	细胞膜 Cell membrane
草莓	FvNCL-1	FvH4_3g18760	578	5.75	63.67	fvb3	细胞膜 Cell membrane
Fragaria vesca	FvNCL-2	FvH4_3g18770	580	5.41	63.51	fvb3	细胞膜 Cell membrane
	FvNCL-3	FvH4_6g26990	580	4.81	63.58	fvb3	细胞膜 Cell membrane
桃	PpNCL-1	Prupe.3G125200	575	5.56	63.45	pp03	细胞膜 Cell membrane
Prunus persica	PpNCL-2	Prupe.4G171300	593	5.39	64.77	pp04	细胞膜 Cell membrane
	PpNCL-3	Prupe.4G171400	579	5.30	63.26	pp04	细胞膜 Cell membrane
白梨	PbNCL-1	Pbr038243.1	585	5.05	63.61	Chr11	细胞膜 Cell membrane
Pyrus bretschneideri	PbNCL-2	Pbr022502.1	585	5.23	64.46	Chr17	细胞膜 Cell membrane
	PbNCL-3	Pbr002291.1	549	5.26	60.01	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-4	Pbr002292.1	580	5.18	63.69	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-5	Pbr002293.1	580	5.18	63.69	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-6	Pbr002294.1	549	5.26	60.01	scaffold1094.0	细胞膜 Cell membrane

物种 Species	基因名称 Gene name	基因登录号 Gene ID	氨基酸 aa	等电点 pI	分子量/kD Molecular weight	染色体 Chromosome	亚细胞定位 Subcellular localization
苹果	MdNCL-1	MD03G1238600	586	5.39	63.75	Chr3	细胞膜 Cell membrane
Malus × domestica	MdNCL-2	MD03G1238700	600	5.30	66.03	Chr3	细胞膜 Cell membrane
	MdNCL-3	MD11G1258400	584	5.01	63.26	Chr11	细胞膜 Cell membrane
	MdNCL-4	MD17G1233800	585	5.34	64.56	Chr17	细胞膜 Cell membrane
拟南芥 Arabidopsis thaliana	AtNCL	AT1G53210.1	585	5.15	63.42	Chr1	细胞膜 Cell membrane
葡萄	VvNCL-1	VIT_212s0028g02570	577	5.73	62.98	Chr12	细胞膜 Cell membrane
Vitis vinifera	VvNCL-2	VIT_219s0014g01740	579	5.21	63.10	Chr19	细胞膜 Cell membrane
草莓	FvNCL-1	FvH4_3g18760	578	5.75	63.67	fvb3	细胞膜 Cell membrane
Fragaria vesca	FvNCL-2	FvH4_3g18770	580	5.41	63.51	fvb3	细胞膜 Cell membrane
	FvNCL-3	FvH4_6g26990	580	4.81	63.58	fvb3	细胞膜 Cell membrane
桃	PpNCL-1	Prupe.3G125200	575	5.56	63.45	pp03	细胞膜 Cell membrane
Prunus persica	PpNCL-2	Prupe.4G171300	593	5.39	64.77	pp04	细胞膜 Cell membrane
	PpNCL-3	Prupe.4G171400	579	5.30	63.26	pp04	细胞膜 Cell membrane
白梨	PbNCL-1	Pbr038243.1	585	5.05	63.61	Chr11	细胞膜 Cell membrane
Pyrus bretschneideri	PbNCL-2	Pbr022502.1	585	5.23	64.46	Chr17	细胞膜 Cell membrane
	PbNCL-3	Pbr002291.1	549	5.26	60.01	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-4	Pbr002292.1	580	5.18	63.69	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-5	Pbr002293.1	580	5.18	63.69	scaffold1094.0	细胞膜 Cell membrane
	PbNCL-6	Pbr002294.1	549	5.26	60.01	scaffold1094.0	细胞膜 Cell membrane