大麦NRAMP全基因组鉴定及重金属胁迫下基因表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1006

摘要/Abstract

摘要：

自然抗性相关巨噬细胞蛋白（natural resistance associated macrophage protein,NRAMP）是植物中一类重要的膜转运蛋白,在转运和重新利用重金属离子中起着重要作用。研究Mn²⁺、Cd²⁺、Cu²⁺三种重金属离子与NRAMP家族基因表达调控之间的作用机制,为进一步明确NRAMP家族基因对大麦抗重金属胁迫的作用提供依据。利用生物信息学的方法从大麦（Hordeum vulgare L.）全基因组中筛选鉴定NRAMP家族基因序列,对该家族成员进行基因结构、保守基序、进化特征及表达谱分析,并利用qRT-PCR的方法检测不同重金属离子胁迫下大麦幼苗中NRAMP基因的表达情况。结果表明,从大麦基因组中共鉴定出10个NRAMP家族成员,且主要定位在细胞膜上;进化分析表明,亲缘关系近的家族成员保守基序、基因结构都更为相似,且大麦与水稻的NRAMP家族成员间亲缘关系更接近;定量检测结果显示,在Mn²⁺胁迫下,HvNramp1、HvNramp2表达水平显著上调;在Cd²⁺胁迫下,除HvNramp8表达量显著下调外,其余基因均上调;在Cu²⁺胁迫下,除HvNramp3外其余6个基因的表达量均显著上调。大麦NRAMP家族基因能够通过正向调控响应重金属离子的胁迫。

关键词: 大麦, NRAMP基因家族, 重金属胁迫, 生理指标, 基因表达

Abstract:

Natural resistance associated macrophage protein（NRAMP）is an important type of membrane transporter in plants,it plays an important role in transporting and reusing heavy metal ions. Studying the mechanism of NRAMP family gene expression regulation under Mn²⁺,Cd²⁺ and Cu²⁺ stress would provide a theoretical basis for further revealing the roles of the NRAMP family genes against heavy metal stress. Bioinformatics methods were used to screen and identify NRAMP family gene sequences from the whole genome of Hordeum vulgare L.,analyzes the gene structure,conserved motifs,evolutionary characteristics and expression profiles of its family members. Moreover,qRT-PCR method was applied to detect the expression of the NRAMP gene in barley seedlings under different heavy metal ion stress. The results showed that there were 10 NRAMP family members in the barley genome,which were mainly located on the cell membrane. Phylogenetic analysis demonstrated that the conserved motifs and gene structures of closely related family members were similar,and the genetic relationship between barley and rice NRAMP family members was closer. The results of quantitative analysis showed that the expression levels of HvNramp1 and HvNramp2 were significantly up-regulated under Mn²⁺ stress. Under Cd²⁺stress,all genes’ expression levels were up-regulated except HvNramp8. Under Cu²⁺ stress,the expression levels of the remaining 6 genes except HvNramp3 were significantly up-regulated. The above results indicate that the NRAMP family genes can respond to the stress of heavy metal ions through positive regulation.

Key words: barley, NRAMP gene family, heavy metal stress, physiological indexes, gene expression

李一涵, 于浪柳, 李春燕, 张蒙蒙, 张晓勤, 方云霞, 薛大伟. 大麦NRAMP全基因组鉴定及重金属胁迫下基因表达分析[J]. 生物技术通报, 2022, 38(6): 103-111.

LI Yi-han, YU Lang-liu, LI Chun-yan, ZHANG Meng-meng, ZHANG Xiao-qin, FANG Yun-xia, XUE Da-wei. Whole Genome Identification of Barley NRAMP and Gene Expression Analysis Under Heavy Metal Stress[J]. Biotechnology Bulletin, 2022, 38(6): 103-111.

图/表 7

表1 荧光定量PCR扩增引物

Table 1 Primers for qRT- PCR analysis

基因 Gene	基因ID Gene ID	引物 Primer	正向引物序列 Forward primer sequence（5'-3'）	反向引物序列 Reverse primer sequence（5'-3'）
HvNramp1	HORVU3Hr1G042440	HvqRT1	CTACCTTCGTGAGTTGGCTTGTA	TGTTGTCTTGTATTGGCACCTCT
HvNramp2	HORVU4Hr1G013220	HvqRT2	AGCACCATAACCGGGACCTA	TGAGCCACTCGTTGAGGACAT
HvNramp3	HORVU4Hr1G069320	HvqRT3	TGTTTACCTCGCCTTCATCGT	CTACCCACTTCCGCTCATCTT
HvNramp4	HORVU4Hr1G077510	HvqRT4	AGGACCTCGCAGACATACCG	AGAAGGACGGATAAAATGCTACG
HvNramp5	HORVU4Hr1G090460	HvqRT5	GGTCTCCAAGCTGGGATGTCT	ATGGCTGTGATAAAATCATCCTG
HvNramp6	HORVU5Hr1G018850	HvqRT6	AACCGAGCCGTACAATGGAC	AGATGATCGTGGCGATACGC
HvNramp7	HORVU5Hr1G050310	HvqRT7	CTGCATAGCAGGTCTCGCACT	CATTTCCACCCAGAAGTGCC
HvNramp8	HORVU7Hr1G078330	HvqRT8	GTGAGGTGAACACTGGCCAGA	CCACCATTATGCCTAAAGAGCC
HvNramp9	HORVU5Hr1G050330	HvqRT9	ATCGGGATAAACGTCTACTTCCT	ATGACAGAGGCGACGTAGAGC
HvNramp10	HORVU7Hr1G106570	HvqRT10	GTGCTAACATGGGCGATCG	ACGGCAAGGTACACTTCCTGTT
HvActin	HORVU1Hr1G002840	Actin	TGGATCGGAGGGTCCATCCT	GCACTTCCTGTGGACGATCGCTG

图1 重金属离子胁迫处理对大麦幼苗叶片生理指标的影响 *和**分别表示重金属离子胁迫处理与对照组之间显著差异或极显著差异,差异显著性统计方法为独立样本t检验（*P<0.05,**P<0.01）,误差棒表示标准误差

Fig. 1 Effects of heavy metal ion stress on the physiological indexes of barley seedling leaves * and ** indicate significant or extremely significant differences between the heavy metal ion stress treatment and the control group,respectively. The independent sample t-test（*P<0.05,**P<0.01）was used for differences analysis,and error bars indicate standard errors

表2 大麦NRAMP蛋白理化性质及染色体定位

Table 2 Physicochemical properties and chromosome location of NRAMP protein in barley

基因 Gene	蛋白质 Protein	氨基酸长度 Amino acid length/aa	分子质量 Molecular mass/Da	理论等电点 Theoretical isoelectric point	亚细胞定位 Subcellular localization	跨膜结构域 Transmembrane domain	染色体定位 Chromosomal localization	物理图谱位置 Physical map location/Mb
HvNramp1	A0A287KVZ6	606	66 306.78	7.82	细胞膜Cell membrane	12	Chr.3H	256.8
HvNramp2	A0A287N9P9	549	60 120.01	5.07	细胞膜Cell membrane	10	Chr.4H	43.2
HvNramp3	M0XUV1	522	56 885.5	5.76	细胞膜Cell membrane	10	Chr.4H	543.5
HvNramp4	M0X4N6	542	58 612.97	7.03	细胞膜Cell membrane	11	Chr.4H	576.7
HvNramp5	A0A287Q4H3	212	22 845.85	5.17	细胞膜Cell membrane	5	Chr.4H	615.3
HvNramp6	F2EHM2	547	58 995.65	5.2	细胞膜Cell membrane	11	Chr.5H	75.1
HvNramp7	A0A287R642	248	58 995.65	5.41	细胞膜Cell membrane	7	Chr.5H	373.2
HvNramp8	A0A287X031	515	55 875.86	8.12	细胞核Nucleus	5	Chr.7H	438.5
HvNramp9	A0A287R6C2	1 010	110 400.77	6.13	细胞膜Cell membrane	11	Chr.5H	373.2
HvNramp10	M0W7C8	548	59 571.99	7.99	细胞膜Cell membrane	12	Chr.7H	591.4

图2 大麦NRAMP基因家族蛋白保守基序、基因结构及系统进化分析 A：大麦NRAMP家族蛋白保守性基序分布;B：大麦、水稻、拟南芥NRAMP基因家族系统进化树。大麦NRAMP系统进化分析,不同颜色的分支代表不同亚家族（I-V）,红色标记为HvNramp;C：大麦NRAMP基因内含子和外显子结构

Fig. 2 Conserved sequences,gene structure and phylogenetic analysis of barley NRAMP gene family A：Barley NRAMP family protein conservative sequence distribution. B：Barley,rice and Arabidopsis NRAMP gene family system evolution tree. Barley NRAMP system evolution analysis,different color branches represent different subfamilies（I-V）,red labeled as HvNramp. C：Intron and exon structure of barley NRAMP gene

图3 大麦NRAMP基因的表达谱分析 EMB：胚胎组织;ROO1：10 cm根组织;LEA：叶组织;INF2：花序组织;NOD：第三茎节间;CAR5：开花后5 d颖果;CAR15：开花后15 d颖果;ETI：黄化叶片;LEM：花序外稃;LOD：花序浆片;PAL：花序内稃;EPI：条纹化表皮;RAC：花序轴;ROO2：28 d根组织;SEN：老叶

Fig. 3 Expression profile analysis of NRAMP gene family in barley EMB：Embryonic tissue. ROO1：10 cm root tissue. LEA：Leaf tissue. INF2：Inflorescence tissue. NOD：Internode of the third stem. CAR5：Caryopsis 5 d after flowering. CAR15：Caryopsis 15 d after flowering. ETI：Etching leaves. LEM：Inflorescence lemma. LOD：Inflorescence lodicule. PAL：Inflorescence lemma. EPI：Striped epidermis. RAC：Inflorescence rachis. ROO2：28 d root tissue. SEN：Old leaves

图4 大麦NRAMP家族基因在染色体上的分布

Fig. 4 Distribution of barley NRAMP family genes on chromosomes

图5 3种重金属离子胁迫下7个大麦NRAMP家族基因相对表达分析

Fig. 5 Relative expression analysis of seven barley NRAMP family genes under three heavy metal ion stresses

参考文献 31

[1]	李洋, 于丽杰, 金晓霞. 植物重金属胁迫耐受机制[J]. 中国生物工程杂志, 2015, 35(9):94-104.
	Li Y, Yu LJ, Jin XX. Mechanism of heavy metal tolerance stress of plants[J]. China Biotechnol, 2015, 35(9):94-104.
[2]	Fang Y, Deng X, Lu X, et al. Differential phosphoproteome study of the response to cadmium stress in rice[J]. Ecotoxicol Environ Saf, 2019, 180:780-788. doi: 10.1016/j.ecoenv.2019.05.068 URL
[3]	吕夏晨, 徐玲, 张蓝天, 等. 褪黑素对干旱胁迫下大麦生理及蜡质基因表达的影响[J]. 植物生理学报, 2020, 56(5):1073-1080.
	Lü XC, Xu L, Zhang LT, et al. Effects of exogenous melatonin on physiology and waxy genes expression in barley under drought stress[J]. Plant Physiol J, 2020, 56(5):1073-1080.
[4]	Skamene E, Pietrangeli CE. Genetics of the immune response to infectious pathogens[J]. Curr Opin Immunol, 1991, 3(4):511-517. pmid: 1755977
[5]	陈可欣, 蒋贤达, 朱祝军, 等. 植物Nramp家族参与金属离子吸收和分配的研究进展[J]. 植物生理学报, 2020, 56(3):345-355.
	Chen KX, Jiang XD, Zhu ZJ, et al. Advances in the study of plant Nramp family involved in metal ion absorption and distribution[J]. Plant Physiol J, 2020, 56(3):345-355. doi: 10.1104/pp.56.3.345 URL
[6]	Peris-Peris C, Serra-Cardona A, Sánchez-Sanuy F, et al. Two NRAMP6 isoforms function as iron and manganese transporters and contribute to disease resistance in rice[J]. Mol Plant Microbe Interact, 2017, 30(5):385-398. doi: 10.1094/MPMI-01-17-0005-R URL
[7]	Cailliatte R, Schikora A, Briat JF, et al. High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions[J]. Plant Cell, 2010, 22(3):904-917. doi: 10.1105/tpc.109.073023 URL
[8]	Takahashi R, Ishimaru Y, Senoura T, et al. The OsNRAMP1 iron transporter is involved in Cd accumulation in rice[J]. J Exp Bot, 2011, 62(14):4843-4850. doi: 10.1093/jxb/err136 pmid: 21697258
[9]	Mäser P, Thomine S, Schroeder JI, et al. Phylogenetic relationships within cation transporter families of Arabidopsis[J]. Plant Physiol, 2001, 126(4):1646-1667. pmid: 11500563
[10]	胡润芳, 林国强, 张广庆, 等. 大豆Nramp基因家族的序列特征与亲缘关系分析[J]. 分子植物育种, 2011, 9(2):245-250.
	Hu RF, Lin GQ, Zhang GQ, et al. Analysis on soybean Nramp gene family sequence features and phylogeny[J]. Mol Plant Breed, 2011, 9(2):245-250.
[11]	卢婷婷, 李艳玲, 李存法, 等. 甘草NRAMP基因家族的鉴定和生物信息学分析[J]. 河南农业科学, 2019, 48(2):40-47.
	Lu TT, Li YL, Li CF, et al. Identification and bioinformatics analysis of NRAMP gene family in Glycyrrhiza uralensis[J]. J Henan Agric Sci, 2019, 48(2):40-47.
[12]	Zhang XQ, Tong T, Tian B, et al. Physiological, biochemical and molecular responses of barley seedlings to aluminum stress[J]. Phyton, 2019, 88(3):253-260. doi: 10.32604/phyton.2019.06143 URL
[13]	徐晓阳, 李国龙, 孙亚卿, 等. 甜菜NAC转录因子鉴定及其在水分胁迫下的表达分析[J]. 植物生理学报, 2019, 55(4):444-456.
	Xu XY, Li GL, Sun YQ, et al. Identification of NAC transcription factors in sugar beet and their expression analyses under water stress[J]. Plant Physiol J, 2019, 55(4):444-456.
[14]	Mayer KF, Waugh R, Brown JW, et al. A physical, genetic and functional sequence assembly of the barley genome[J]. Nature, 2012, 491(7426):711-716. doi: 10.1038/nature11543 URL
[15]	Zhang X, Zhang LT, Chen YY, et al. Genome-wide identification of the SOD gene family and expression analysis under drought and salt stress in barley[J]. Plant Growth Regul, 2021, 94(1):49-60. doi: 10.1007/s10725-021-00695-8 URL
[16]	Mani A, Sankaranarayanan K. In silico analysis of natural resistance-associated macrophage protein(NRAMP)family of transporters in rice[J]. Protein J, 2018, 37(3):237-247. doi: 10.1007/s10930-018-9773-y URL
[17]	王利华. 拟南芥AtNRAMP3转运蛋白的结构与功能研究[D]. 南京: 南京农业大学, 2017:1-74.
	Wang LH.Structure and function of AtNRAMP 3 transporter in Arabidopsis thaliana[D]. Nanjing: Nanjing Agricultural University, 2017:1-74.
[18]	胡阳阳, 孙琳琳, 王多祥, 等. 水稻NRL基因家族的全基因组鉴定与生物信息学分析[J]. 分子植物育种, 2020. https://kns.cnki.net/kcms/detail/46.1068.S.20200904.1146.006.html.
	Hu YY, Sun LL, Wang DX, et al. Genome-wide identification and bioinformatics analysis of NRL gene family in rice[J]. Mol Plant Breed, 2020. https://kns.cnki.net/kcms/detail/46.1068.S.20200904.1146.006.html.
[19]	杨猛. 水稻NRAMP家族基因在Mn和Cd转运中的功能研究[D]. 武汉: 华中农业大学, 2014:1-139.
	Yang M. Functional analysis of rice NRAMP genes in Mn and Cd transport[D]. Wuhan: Huazhong Agricultural University, 2014:1-139.
[20]	Zhang XQ, Chen HN, Jiang H, et al. Measuring the damage of heavy metal cadmium in rice seedlings by SRAP analysis combined with physiological and biochemical parameters[J]. J Sci Food Agric, 2015, 95(11):2292-2298. doi: 10.1002/jsfa.6949 URL
[21]	Yuchun RAO, Ran JIAO, Sheng WANG, et al. SPL36 encodes a receptor-like protein kinase that regulates programmed cell death and defense responses in rice[J]. Rice:N Y, 2021, 14(1):34.
[22]	何凤, 刘攀峰, 王璐, 等. 干旱胁迫及复水对杜仲苗生理特性的影响[J]. 植物生理学报, 2021, 57(3):661-671.
	He F, Liu PF, Wang L, et al. Effect of drought stress and rewatering on physiological characteristics of Eucommia ulmoides seedling[J]. Plant Physiol J, 2021, 57(3):661-671.
[23]	Bairoch A. The PROSITE dictionary of sites and patterns in proteins, its current status[J]. Nucleic Acids Res, 1993, 21(13):3097-3103. pmid: 8332530
[24]	刘营, 尹泽, 江姚兰, 等. 甘蔗ScNRAMP基因家族的全基因组鉴定与生物信息学分析[J/OL]. 广西植物, 2021. DOI: 10.11931/guihaia.gxzw202104005. doi: 10.11931/guihaia.gxzw202104005
	Liu Y, Yin Z, Jiang YL, et al. Identification and bioinformatics analysis of ScNRAMP gene family in sugarcane[J/OL]. Guihaia, 2021. DOI: 10.11931/guihaia.gxzw202104005. doi: 10.11931/guihaia.gxzw202104005
[25]	Gao H, Xie W, Yang C, et al. NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency[J]. New Phytol, 2018, 217(1):179-193. doi: 10.1111/nph.14783 URL
[26]	Lanquar V, Ramos MS, Lelièvre F, et al. Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency[J]. Plant Physiol, 2010, 152(4):1986-1999. doi: 10.1104/pp.109.150946 URL
[27]	Thomine S, Wang R, Ward JM, et al. Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes[J]. PNAS, 2000, 97(9):4991-4996. pmid: 10781110
[28]	Cellier M, Govoni G, Vidal S, et al. Human natural resistance-associated macrophage protein:cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression[J]. J Exp Med, 1994, 180(5):1741-1752. doi: 10.1084/jem.180.5.1741 pmid: 7964458
[29]	Ishimaru Y, Bashir K, Nakanishi H, et al. OsNRAMP5, a major player for constitutive iron and manganese uptake in rice[J]. Plant Signal Behav, 2012, 7(7):763-766. doi: 10.4161/psb.20510 URL
[30]	李亚敏, 巩宗强, 贾春云, 等. 玉米镉转运基因ZmNramp1的鉴定及对镉胁迫的响应[J]. 生态学杂志, 2021, 40(7):2016-2023.
	Li YM, Gong ZQ, Jia CY, et al. Identification of the cadmium transport gene ZmNramp1 in maize and its response to cadmium stress[J]. Chin J Ecol, 2021, 40(7):2016-2023.
[31]	孙丽娟, 程龙军. 缺Cu²⁺和Zn²⁺对水稻OsNRAMP家族基因表达与主要金属离子吸收的影响[J]. 植物生理学报, 2011, 47(9):879-884.
	Sun LJ, Cheng LJ. Effects of deficiency of copper and zinc on OsNRAMPs expression and main metal-ions uptake in Oryza sativa L[J]. Plant Physiol J, 2011, 47(9):879-884.