南方型紫花苜蓿叶片盐胁迫应答基因鉴定与分析

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (1471 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要以南方型紫花苜蓿(Medicago sativa ‘Millennium’)为材料，以正常培养(WT_CK2)和盐胁迫(WT_N2)条件下的2个样品叶片进行转录组分析，鉴定紫花苜蓿叶片盐胁迫应答相关基因，从转录组水平揭示南方型紫花苜蓿适应盐胁迫环境的分子机制。提取常培养(WT_CK2)和盐胁迫(WT_N2)叶片总RNA，以Illumina Hiseq 2000 平台进行RNA Sequencing(RNA-Seq)分析。同时，对上调和下调DEGs(differential-expressed genes)进行基因本体(Gene Ontology, GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)代谢途径富集分析。选择DEGs 6个，以 qRT-PCR验证RNA-Seq 结果的可靠性。结果表明，滤低质量reads后，WT_CK2和WT_N2分别保留了62 771 040和60 394 756 对高质量reads 用于差异表达基因的筛选，其中52.47%和53.07%的reads 能准确比对到参考序列蒺藜苜蓿(Medicago truncatula)上，说明RNA-Seq 结果和参考序列可靠。DEGs 鉴定结果表明，7 497个基因受盐胁迫诱导差异表达，其中上调表达4 078个，3 419下调表达，其中包括46个转录因子家族的474个转录因子基因在盐胁迫差异表达。GO富集分析表明，差异表达基因广泛涉及结合、催化活性、细胞过程、代谢过程和刺激响应。KEGG代谢分析表明，差异表达基因广泛涉及次生代谢、代谢途径、苯丙素的生物合成、黄酮类生物合成和植物激素信号转导。紫花苜蓿叶片盐胁迫响应中活性氧清除类、渗透调节和离子转运类基因上调，参与生物合成和钙依赖蛋白激酶信号转导途径，丝裂原活化蛋白激酶，PP2C(protein phosphatase 2C)基因家族、ABA(abscisic acid)通路和激素(水杨酸, 乙烯和茉莉酸)信号途径基因表达上调，C3H、NAC、WRKY 和AP2/EREBP转录家族因子不同程度上调，而基础代谢和蛋白合成途径总体上受到抑制。此外，候选了与盐胁迫响应相关的海藻糖-6-磷酸合成酶基因、Na+/H+逆向转运蛋白基因、类受体激酶基因、钙依赖性蛋白激酶基因、非酵解型蛋白激酶基因、类萌发素蛋白基因及相关转录因子基因等。qRT-PCR 检测6 个DEGs 的表达模式与RNA-Seq 分析结果一致，证实了RNA-Seq 结果的可靠性。本研究为阐明南方型紫花苜蓿耐盐分子机制提供基础资料。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	马进
	郑钢

关键词 ：紫花苜蓿, 基因, 叶片转录组, 盐胁迫

Abstract：In order to investigate the molecular mechanism of salt tolerance, the transcriptomes of southern type Alfalfa (Medicago sativa ‘Millenium’) under control condition (WT_CK2) were compared with those under NaCl-treated (WT_CK2) condition. Total RNA was extracted from leaves under control and NaCl-treated conditions, and then was used for RNA Sequencing (RNA-Seq) analysis on the Illumina Hiseq 2000 platform. Meanwhile, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on the differentially expressed genes (DEGs). qRT-PCR technique was used to validate the expression data of 6 randomly selected DEGs. The results indicated that after filtration of low-quality reads, a total of 62 771 040 and 60 394 756 high-quality reads were acquired, respectively, in control and NaCl-treated samples for the identification of DEGs. Among these high-quality reads, 52.47% (control) and 53.07% (NaCl-treated) could be accurately mapped to the reference genome of Medicago truncatula, thus validating the RNA-Seq data and the reference sequence. A total of 7 497 DEGs were identified, among which 4 078 and 3 419 were detected to be up-and down-regulated, respectively, under NaCl treatment. These genes include 474 transcription factors (TFs) from 46 TF families. GO enrichment analysis showed these DEGs were involved in pathways like binding, catalytic activity, cellular process, metabolic process and response to stimulus. KEGG pathway analysis showed that these DEGs were involved in biosynthesis of secondary metabolites, metabolic pathways, phenylpropanoid biosynthesis, favonoid biosynthesis and plant hormone signal transduction. Important DEGs contained genes involved in osmotic adjustment, ion transport, and pathways involved in biosynthesis and signal transduction. Genes coding calcium-dependent protein kinase, mitogen-activated protein kinase, PP2C family, ABA pathway and hormones (salicylic acid, ethylene and jasmonic acid) signal pathways were up-regulated, TFs from C3H, NAC, WRKY and AP2/EREBP TF families were also differentially up-regulated. On the other hand, genes related to fundamental metabolic mechanism and protein synthesis were generally down-regulated under salt stress. In addition, we identified many candidate genes, such as trehalose-6-phosphate synthase gene, vacuolar Na+/H+ antiporter gene, stress-induced protein kinase gene, calcium-dependent protein kinase gene, sucrose non-fermenting 1-related protein kinase, SnRK, germin-like protein gene, to be related to salt tolerance. Expression analysis of the 6 DEGs with qRT-PCR was in consistent with those of RNA-Seq data, thus furtherly confirming the reliability of RNA-Seq results. This research provides the basic evidence for the molecular mechanisms of salt tolerance in southern type alfalfa.

Key words： Medicago sativa ‘Millenium’ Gene Leaf transcriptome Salt stress

收稿日期: 2015-05-04 出版日期: 2015-10-11

基金资助:南方型紫花苜蓿盐诱导蛋白基因克隆及功能分析

通讯作者: 马进 E-mail: majinzjl@163.com

引用本文:

马进郑钢. 南方型紫花苜蓿叶片盐胁迫应答基因鉴定与分析[J]. , 2015, 23(12): 1531-1541.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I12/1531

郭鹏，邢鑫，张万筠，等. 紫花苜蓿盐诱导类受体蛋白激酶基因MsSIK1的克隆及功能分析 [J]. 中国农业科学，2014，47(23)：4573–4581.（GUO PENG，XING XING，ZHANG WANJUN，et al. Cloning and function analysis of a salt-stress-induced receptor like protein kinase gene MsSIK1 from alfalfa [J]. Scientia Agricultura Sinica，2014，47(23):4573–4581.）
李明娜, 龙瑞, 杨青川, 等. 紫花苜蓿盐诱导HD-Zip类转录因子MsHB2的克隆及功能分析 [J].中国农业科学，2014，47(4)：622–632.（LI MINGNA，LONG RUI，YANG QINGCHUAN，et al. Cloning and function analysis of a salt-stress-induced HD-Zip transcription factor MsHB2 from alfalfa [J]. Scientia Agricultura Sinica, 2014，47(4):622–632.）
马进, 郑钢, 蔡建国.2011. 南方型紫花苜蓿基因型在组织培养条件下对NaCl的耐性[J].浙江农业学报, 23(4): 782－786.（Ma J, Zheng G, Cai J G. 2011. NaCl tolerance of different genotypes of southern type of Medicago sativa under tissue culture. Acta Agriculturae Zhejiangensis, 2011, 23( 4): 782－786）
Asano T, Hakata M, Nakamura H, et al. 2011 .Functional characterization of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice [J]. Plant Molecular Biology, 75(1–2): 179–191.
Banerjee J, Das N, Dey P, et al. 2010. Transgenically expressed rice germin-like protein1 in tobacco causes hyper-accumulation of H2O2 and reinforcement of the cell wall components [J]. Biochemical and Biophysical Research Communications, 402(4): 637–643.
Bonawitz N D, Soltau W L, Blatchley M R, et al. 2012. REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis [J]. Journal of Chemical Biology, 287(8):5434–5445.
Burnette R N, Gunesekera B M, Gillaspy G E. 2013 . An Arabidopsis inositol 5-phosphatase gain-of-function alters abscisic acid signaling [J]. Plant Physiology, 132(2): 1011–1019.
Chaudhary M T, Wainwright S J, Merrett M J. 1996.Comparative NaCl tolerance of lucerne regenerated from salt-selected suspension cultures[J]. Plant Science, 9(114): 221–223.
Chen L J, Wuriyanghan H, Zhang Y Q, et al. 2013. An S-domain receptor-like kinase,OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice [J]. Plant Physiolology, 163(4): 1752–1765.
Chitteti B R, Peng Z H. 2007 .Proteome and phosphoproteome differential expression under salinity stress in rice (Oryza sativa) roots[J]. Journal of Proteome Research, 6(5): 1718–1727.
Deinlein U, Stephan A B, Horie T, et al. 2014 .Plant salt-tolerance mechanisms [J]. Trends in Plant Science, 19(6): 371–379.
Geilfus C M, Z?rb C, Mühling K H. 2010. Salt stress differentially affects growth-mediating β-expansins in resistant and sensitive maize (Zea mays L.) [J]. Plant Physiology and Biochemistry, 48: 993–998.
Ginzberg I, Stein H, Kapulnik Y. 1998. Isolation and characterization of two different Cdnas of Delta(1)-pyrroline 5-carboxylate synthase in alfalfa, trascriptionally induced upon salt stress [J].Plant Molecular Biology, 38(5): 755–764.
Grover, A. 2012. Plant chitinases: genetic diversity and physiological roles [J]. Critical Reviews in Plant Sciences，31:57–73
Jiang Y Q, Yang B, Harris N S, et al. 2007 .Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots [J]. Journal of Experimental Botany, 58(13): 3591–3607.
Luo Y, Liu Y B, Dong Y X, et al. 2009. Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment [J].Journal Plant Physiology, 166(4): 385–394.
Ma L C, Wang Y R, Liu W X, et al. 2014. Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid, drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate accumulation [J].Biotechnology Letters, 36(11): 2331–2341.
Miller G, Suzuki N, Ciftci-Yilmaz S, et al. 2010 . Reactive oxygen species homeostasis and signalling during drought and salinity stresses [J]. Plant Cell and Environment, 33(4): 453–467.
M?llerl S, Gilliham M, Jha D, et al. 2009. Shoot Na+ exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na+ transport in Arabidopsis [J]. Plant Cell, 21(7):2163-2178.
Rouhier N, Couturier J, Jacquot J P. 2006 . Genome-wide analysis of plant glutaredoxin systems [J]. Journal of Experimental Botany, 57(8): 1685–1696.
Roose J L, Wegener K M, Pakrasi H B. 2007. The extrinsic proteins of PhotosystemⅡ[J]. Photosynthesis Research，92(3): 369–387.
Soon F F, Ng L M, Zhou X E, et al. 2012. Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases [J]. Science, 2012, 335(6064): 85–-88.
Van Damme E J, Barre A, Rougé P, et al. 2004. Cytoplasmic/ nuclear plant lectins: A new story [J].Trends in Plant Science, 9: 484–489.
Winicov I, Shizadegan M. 1997. Tissue speicific modulation of salt inducible gene expression callus versus whole plant response in salt tolerance alfalfa [J].Physiol Planta, 100(2): 314–319.
Wu T M, Lin W R, Kao Y T, et al. 2013. Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice [J]. Plant Molecular Biology, 83(4-5): 379–390.
Xiang Y, Huang Y M, Xiong L Z. 2007. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. [J]. Plant Physiology, 144(3): 1416–1428.
Xie Z M, Zou H F, Lei G, et al. 2009. Soybean trihelix transcription factors GmGT-2A and GmGT-2B improve plant tolerance to abiotic stresses in transgenic Arabidopsis [J]. PLoS One, 2009, 4(9): e6898.
Yadav D K, Tuteja N. 2011. Rice G-protein coupled receptor (GPCR):in silico analysis and transcription regulation under abiotic stress[J]. Plant Signal and Behavior, 6(8): 1079–1086.
Yamaguchi T, Hamamoto S, Uozumi N. 2013. Sodium transport system in plant cells [J]. Front Plant Science, 2013, 4: 410.
Zahaf O, Blanchet S, Zelicourt A, et al. 2012. Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncateula genotypes. Molecular Plant, 5(5):1068–1081.
Zhang H, Han B, Wang T, et al. 2012. Mechanisms of plant salt response: insights from proteomics [J]. Journal of Proteome Research, 11(1): 49–67.
Zhao L, Gu R L, Gao P, et al. 2008. A nonsymbiotic hemoglobin gene from maize, ZmHb, is involved in response to submergence, high-salt and osmotic stresses [J]. Plant Cell Tissue and Organ Culture, 95: 227–237.