铝诱导的茶树根系转录组变化分析

doi:10.13305/j.cnki.jts.2019.05.002

摘要/Abstract

摘要： 探讨茶树响应铝（Aluminum,Al）的基因调控网络和表达模式,确定一些关键候选基因,为茶树耐Al分子机制研究奠定基础。测定了0、0.2、1、2、4βmmol·L^-1 5个Al³⁺浓度处理7βd的福鼎大白茶根系抗氧化酶活性和Al含量变化,并提取0βmmol·L^-1（R0）、1βmmol·L^-1（R1）和4βmmol·L^-1（R4）3个浓度下的茶树根系总RNA,通过Illumina Hiseq Xten平台进行高通量转录组测序。结果表明,随着Al³⁺浓度的升高,根系POD（Peroxidase,过氧化物酶）活性逐渐下降,APX（Ascorbic acid peroxidase,抗坏血酸过氧化物酶）活性则逐渐升高。SOD（Superoxide dismutase,超氧化物歧化酶）活性在Al³⁺浓度为1βmmol·L^-1时最高,CAT（Catalase,过氧化氢酶）活性在各处理间无显著差异。根系中Al含量随着Al³⁺浓度的升高呈先上升后下降趋势,在Al³⁺浓度为1βmmol·L^-1时达到最高。经筛选得到R1 VS R0,R4 VS R0,R4 VS R1的DEGs（Differentially expressed genes）分别为1β894、2β439个和1β384个,显著上调（下调）的差异表达基因分别有733（1β161）、846（1β593）个和628（756）个。GO富集分析表明,3个处理组在生物学途径中富集最多的类别均为刺激响应。在分子功能和细胞组件方面,R1 VS R0和R4 VS R0富集最多的类别均为核酸结合转录因子活性和细胞外围,R4 VS R1富集最多的类别为氧化还原酶活性相关基因和膜区域。KEGG富集分析表明,R1 VS R0、R4 VS R0、R4 VS R1分别显著富集了29、41条和19条Pathway,它们包括转录因子、转运蛋白、植物-病原菌互作、苯丙烷生物合成途径等,鉴定到多个参与调控活性氧代谢、有机酸或金属转运蛋白、转录因子及细胞壁结构修饰等生理过程的基因在Al诱导后上调或抑制表达,显示这些基因与茶树耐Al分子机制密切相关。

关键词: 茶树, Al, 根系, 差异表达基因, RNA测序

Abstract: The aim of this study was to investigate the gene regulation network and expression pattern of the response to aluminum (Al) in tea plants, and to identify the key candidate genes for understanding molecular mechanism of Al tolerance in tea plants. The roots’ antioxidant enzyme activities and Al content of Fuding Dabaicha cultivar were detected under 0βmmol·L^-1, 0.2βmmol·L^-1, 1βmmol·L^-1, 2βmmol·L^-1 and 4βmmol·L^-1 Al³⁺ concentrations for 7βd. The total RNA of roots under 0βmmol·L^-1 (R0), 1βmmol·L^-1 (R1) and 4βmmol·L^-1 (R4) Al³⁺ concentrations were extracted for high-through transcriptome sequencing by Illumina Hiseq Xten platform. The results showed that with the increase of Al concentration, POD activity decreased while APX activity increased gradually. SOD activity reached the highest peak at the Al concentration of 1βmmol·L^-1. However, CAT activity showed no significant difference among five treatments. The Al content first increased and then decreased with the increase of Al³⁺ concentration, and reached the highest peak at the Al³⁺ concentration of 1βmmol·L^-1.The DEGs of R1 VS R0, R4 VS R0, R4 VS R1 were 1β894, 2β439 and 1β384 respectively with 733 (1β161), 846 (1β593) and 628 (756) DEGs significantly up-regulated (down-regulated). GO enrichment analysis shows that the most enrichment biological pathway of three samples were all stimulus responses. In terms of molecular function and cell components, R1 VS R0 and R4 VS R0 were mostly enriched in nucleic acid binding transcription factor activity and cell periphery, while R4 vs R1 were mostly enriched in redox enzyme activity and membrane. KEGG enrichment analysis illustrates that they were significantly enriched in 29, 41, and 19 pathways, respectively, including transcription factors, transporters, plant-pathogen interactions, and phenylpropanoid biosynthesis pathways. It was found that genes involved in physiological processes such as reactive oxygen metabolism, organic acids or metal transporters, transcription factors and cell wall structure modification were up-regulated or inhibited after Al induction, suggesting that these genes were closely related to the molecular mechanism of Al tolerance in tea plants.

Key words: Camellia sinensis, aluminum, root, differential expressed genes, RNA sequencing

中图分类号:

S571.1
Q52

黄丹娟, 谭荣荣, 陈勋, 王红娟, 龚自明, 王友平, 毛迎新. 铝诱导的茶树根系转录组变化分析[J]. 茶叶科学, 2019, 39(5): 506-520. doi: 10.13305/j.cnki.jts.2019.05.002.

HUANG Danjuan, TAN Rongrong, CHEN Xun, WANG Hongjuan, GONG Ziming, WANG Youping, MAO Yingxin. Transcriptome Analysis of Root Induced by Aluminum in Tea Plants (Camellia sinensis)[J]. Journal of Tea Science, 2019, 39(5): 506-520. doi: 10.13305/j.cnki.jts.2019.05.002.

参考文献 56

[1]	Ma J F, Chen Z C, Shen R F.Molecular mechanisms of Al tolerance in gramineous plants[J]. Plant and Soil, 2014, 381(1/2): 1-12.
[2]	Hajiboland R, Rad S B, Barcelo J, Poschenrieder C.Mechanisms of aluminum-induced growth stimulation in tea (Camellia sinensis)[J]. Journal of Plant Nutrition and Soil Science, 2013, 176(4): 616-625.
[3]	王敏, 宁秋燕, 石元值. 茶树幼苗对不同浓度铝的生理响应差异研究[J]. 茶叶科学, 2017, 37(4): 337-346.
[4]	Morita A, Yanagisawa O, Maeda S, et al.Tea plant(Camellia sinensis L.) roots secrete oxalic acid and caffeine into medium containing aluminum[J]. Journal of Soil Science and Plant Nutrition, 2011(57): 796-802.
[5]	刘腾腾, 郜红建, 宛晓春, 等. 铝对茶树根细胞膜透性和根系分泌有机酸的影响[J]. 茶叶科学, 2011, 31(5): 458-462.
[6]	疏再发. 根系有机酸和细胞壁果胶甲酯化参与茶树耐铝/解铝毒机制的研究[D]. 南京: 南京农业大学, 2016: 30-31.
[7]	Li D Q, Shu Z F, Ye X L, et al.Cell wall pectin methyl-esterification and organic acids of root tips involve in aluminum tolerance in Camellia sinensis[J]. Plant Physiology and Biochemistry, 2017, 119: 265-274.
[8]	Tolrà R, Vogel-Miku K, Hajiboland R, et al.Localization of aluminium in tea ( Camellia sinensis ) leaves using low energy X-ray fluorescence spectro-microscopy[J]. Journal of Plant Research, 2011, 124(1): 165-172.
[9]	Hajiboland R, Poschenrieder C.Localization and compartmentation of Al in the leaves and roots of tea plants[J]. Phyton, 2015, 84(1): 86-100.
[10]	Gao H J, Zhao Q, Zhang X C, et al.Localization of fluoride and aluminum in subcellular fractions of tea leaves and roots[J]. Journal of Agricultural and Food Chemistry, 2014, 62(10): 2313-2319.
[11]	Panda S K, Sahoo L, Katsuhara M, et al.Overexpression of alternative oxidase gene confers aluminum tolerance by altering the respiratory capacity and the response to oxidative stress in tobacco cells[J]. Molecular Biotechnology, 2013, 54(2): 551-563.
[12]	Larsen P B, Geisler M J, Jones C A, et al.ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis[J]. The Plant Journal, 2005, 41(3): 353-363.
[13]	Larsen P B, Cancel J, Rounds M, et al.Arabidopsis ALS1 encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment[J]. Planta, 2007, 225(6): 1447-1458.
[14]	Arenhart R A, Bai Y, Oliveira L F, et al.New insights into aluminum tolerance in rice: the ASR5 protein binds the STAR1 promoter and other aluminum-responsive genes[J]. Molecular Plant, 2014, 7(4): 709-721.
[15]	Yokosho K, Yamaji N, Ma J F.Global transcriptome analysis of Al-induced genes in an Al-accumulating species, common buckwheat (Fagopyrum esculentum Moench)[J]. Plant and Cell Physiology. 2014, 55(12): 2077-2091.
[16]	Chen H, Lu C P, Jiang H, et al.Global transcriptome analysis reveals distinct aluminum-tolerance pathways in the Al-accumulating species Hydrangea macrophylla and marker identification[J]. PLoS One, 2015, 10(12): e0144927. DOI: 10.1371/journal.pone.0144927.
[17]	Kobayashi Y, Hoekenga O A, Itoh H, et al.Characterization of AtALMT1 expression in aluminum-inducible malate release and its role for rhizotoxic stress tolerance in Arabidopsis[J]. Plant Physiology, 2007, 145(3): 843-852.
[18]	Liu J, Jurandir V, Magalhaes J V, et al.Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance[J]. The Plant Journal. 2009, 57(3): 389-399.
[19]	Yamaji N, Huang C F, Nagao S, et al.A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice[J]. Plant Cell, 2009, 21(10): 3339-3349.
[20]	Sawaki Y, Iuchi S, Kobayashi Y, et al.STOP1 regulates multiple genes that protect Arabidopsis from proton and aluminum toxicities. Plant Physiology[J]. 2009, 150(1): 281-294.
[21]	Sawaki Y, Kobayashia Y, Kihara-Doic T, et al.Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes[J]. Plant Science, 2014, 223: 8-15.
[22]	Huang C F, Yamaji N, Mitani N, et al.A bacterial-type ABC transporter is involved in aluminum tolerance in rice[J]. Plant Cell, 2009, 21(2): 655-667.
[23]	Xu Q S, Wang Y, Ding Z T, et al.Aluminum induced physiological and proteomic responses in tea (Camellia sinensis) roots and leaves[J]. Plant Physiology and Biochemistry, 2017, 115: 141-151.
[24]	Li Y, Huang J, Song X W, et al.An RNA-Seq transcriptome analysis revealing novel insights into aluminum tolerance and accumulation in tea plant[J]. Planta, 2017, 246(1): 91-103.
[25]	Zhao H, Huang W, Zhang Y G, et al.Natural variation of CsSTOP1 in tea plant (Camellia sinensis) related to aluminum tolerance[J]. Plant and Soil, 2018, 431(1/2): 71-87.
[26]	Fan K, Wang M, Gao Y, et al.Transcriptomic and ionomic analysis provides new insight into the beneficial effect of al on tea roots’ growth and nutrient uptake[J]. Plant Cell Reports, 2019, 38(6): 715-729.
[27]	宁秋燕. 茶树根系中XTHs和Expansins对不同浓度铝的响应研究[D]. 北京: 中国农业科学院研究生院, 2018: 29-31.
[28]	曹红利. 茶树bZIP家族基因的非生物胁迫响应及C亚家族CsbZIP6和CsbZIP4的功能初步分析[D]. 北京: 中国农业科学院研究生院, 2016: 16-17.
[29]	Wei C L, Yang H, Wang S B, et al.Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality[J]. Proc Natl Acad Sci USA, 2018, 115(18): 4151-4158.
[30]	Mortazavi A, Williams B A, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nature Methods, 2008, 5(7): 621-628.
[31]	Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the <inline-graphic xlink:href="1000-369X-39-5-506/img_1.wmf"/> Method[J]. Methods, 2001, 25(4): 402-408.
[32]	Mukhopadyay M, Bantawa P, Das A, et al.Changes of growth, photosynthesis and alteration of leaf antioxidative defense system of tea [Camellia sinensis (L.) O. Kuntze] seedlings under aluminum stress[J]. Biometals, 2012, 25(6): 1141-1154.
[33]	Li C L, Xu H M, Xu J, et al.Effects of aluminium on ultrastructure and antioxidant activity in leaves of tea plant[J]. Acta Physiologiae Plantarum, 2011, 33(3): 973-978.
[34]	Richards K D, Schott E J, Sharma Y K, et al.Aluminum induces oxidative stress genes in Arabidopsis thaliana[J]. Plant Physiology, 1998, 116(1): 409-418.
[35]	Chowra U, Yanase E, Koyama H, et al.Aluminium-induced excessive ROS causes cellular damage and metabolic shifts in black gram Vigna mungo (L.) Hepper[J]. Protoplasma, 2017, 254(1): 293-302.
[36]	Ezaki B, Gardner R C, Ezaki Y, et al.Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress[J]. Plant Physiology, 2000, 122(3): 657-665.
[37]	李勇. 茶树响应铝的遗传变异及铝富集候选基因挖掘[D]. 武汉: 华中农业大学, 2017: 36.
[38]	You J F, Zhang H M, Liu N, et al.Transcriptomic responses to aluminum stress in soybean roots[J]. Genome, 2011, 54(11): 923-933.
[39]	Guo P, Qi Y P, Yang L T, et al.Root adaptive responses to aluminum-treatment revealed by RNA-Seq in two Citrus species with different aluminum-tolerance[J]. Front in Plant Science, 2017, 8: 330. DOI: 10.3389/fpls.2017.00330.
[40]	王晓珠, 孙万梅, 马义峰, 等. 拟南芥ABC转运蛋白研究进展[J].植物生理学报, 2017, 53(2): 133-144.
[41]	Li J Y, Liu J, Dong D, et al.Natural variation underlies alterations in Nramp aluminum transporter (NRAT1) expression and function that play a key role in rice aluminum tolerance[J]. Proc Natl Acad Sci USA, 2014, 111(17): 6503-6508.
[42]	Zhang H, Tan Z Q, Hu L Y, et al.Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings[J]. Journal of Integrative Plant Biology, 2010, 52(6): 556-567.
[43]	Dawood M, Cao F, Jahangir M M, et al. Alleviation of aluminum toxicity by hadrogen suilfide is related to elevated ATPase,suppressed aluminum uptake and oxidative stress in barley [J]. Journal of Hazardous Materials, 2012, 209/210: 121-128.
[44]	Guo P, Li Q, Qi Y P, et al.Sulfur-Mediated-Alleviation of Aluminum-Toxicity in Citrus grandis seedlings[J]. International Journal of Molecular Sciences, 2017, 18(12): 2570. DOI: 10.3390/ijms18122570.
[45]	Negishi T, Oshima K, Hattori M, et al.Tonoplast- and plasma membrane-localized aquaporin-family transporters in blue Hydrangea Sepals of aluminum hyperaccumulating plant[J]. PLoS One, 2012, 7(8): e43189. DOI: 10.1371/journal.pone.0043189.
[46]	范伟, 娄和强, 龚育龙, 等. 调控铝诱导根尖有机酸分泌的分子机制[J]. 植物生理学报, 2014, 50(10): 1469-1478.
[47]	Chen L, Song Y, Li S, et al.The role of WRKY transcription factors in plant abiotic stresses[J]. Biochimica et Biophysica Acta, 2012, 1819(2): 120-128.
[48]	Kumari M, Taylor G J, Deyholos K.Transcriptomic responses to aluminum stress in roots of Arabidopsis thaliana[J]. Molecular Genetics and Genomics, 2008, 279(4): 339-357.
[49]	Goodwin S B, Sutter T R.Microarray analysis of Arabidopsis genome response to aluminum stress[J]. Biologia Plantarum, 2009, 53(1): 85-99.
[50]	Ding Z J, Yan J Y, Xu X Y, et al. WRKY46 functions as a transcriptional repressor of ALMT1, regulating aluminum induced malate secretion in Arabidopsis[J]. The Plant Journal, 2013, 76(5): 825-835.
[51]	Horst W J, Wang Y, Eticha D.The role of the root apoplast in aluminium-induced inhibition of root elongation and in aluminium resistance of plants: a review[J]. Annals of Botany, 2010, 106(1): 185-197.
[52]	Teraoka T, Kaneko M, Mori S, et al.Aluminum rapidly inhibits cellulose synthesis in roots of barley and wheat seedlings[J]. Journal of Plant Physiology, 2002, 159(1): 17-23.
[53]	Tabuchi A1, Matsumoto H. Changes in cell-wall properties of wheat (Triticum aestivum) roots during aluminum-induced growth inhibition[J]. Physiology Plant, 2001, 112(3): 353-358.
[54]	Potter I, Fry S C.Changes in xyloglucan endotransglycosylase (XET) activity during hormone-induced growth in lettuce and cucumber hypocotyls and spinach cell suspension cultures[J]. Journal of Experimental Botany, 1994, 45: 1703-1710.
[55]	Chandran D, Sharopova N, Ivashuta S, et al.Transcriptome profiling identified novel genes associated with aluminum toxicity, resistance and tolerance in Medicago truncatula[J]. Planta, 2008, 228(1): 151-166.
[56]	Yang J L, Zhu X F, Peng Y X, et al.Cell wall hemicellulose contributes significantly to aluminum adsorption and root growth in Arabidopsis[J]. Plant Physiology. 2011, 155(4): 1885-1892.