[1]王 影,邱文敏,李 鹤,等.东南景天SaWRKY7基因对镉胁迫的响应研究[J].南京林业大学学报(自然科学版),2019,43(03):059-66.[doi:10.3969/ j.issn.1000-2006.201810007]
 WANG Ying,QIU Wenmin,LI He,et al.Research on the response of SaWRKY7 gene to cadmium stress in Sedum alfredii Hance[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(03):059-66.[doi:10.3969/ j.issn.1000-2006.201810007]
点击复制

东南景天SaWRKY7基因对镉胁迫的响应研究
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
43
期数:
2019年03期
页码:
059-66
栏目:
研究论文
出版日期:
2019-05-15

文章信息/Info

Title:
Research on the response of SaWRKY7 gene to cadmium stress in Sedum alfredii Hance
文章编号:
1000-2006(2019)03-0059-08
作者:
王 影12邱文敏2李 鹤2贺雪莲2刘明英2韩小娇2曲同宝1*卓仁英2*
1.吉林农业大学园艺学院,吉林 长春 130118; 2.中国林业科学研究院亚热带林业研究所,浙江省林木育种重点实验室,浙江 杭州 311400
Author(s):
WANG Ying12 QIU Wenmin2 LI He2 HE Xuelian2 LIU Mingying2 HAN Xiaojiao2 QU Tongbao1* ZHUO Renying2*
1. College of Horticulture, Jilin Agricultural University, Changchun 130118, China; 2 Key Laboratory of Tree Breeding of Zhejiang Province, The Research Institute of Subtropical of Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
关键词:
东南景天 SaWRKY7 转录因子 镉胁迫
Keywords:
Sedum alfredii Hance SaWRKY7 transcription factor cadmium stress
分类号:
Q945.78
DOI:
10.3969/ j.issn.1000-2006.201810007
文献标志码:
A
摘要:
【目的】镉对植物具有高毒性,WRKY转录因子广泛参与植物逆境胁迫。研究镉诱导下WRKY基因的胁迫响应和抗性机制。【方法】以超积累型东南景天为试材,通过PCR克隆得到1个东南景天WRKY转录因子家族基因序列,结合生物信息学方法,分析其蛋白质结构和亲缘关系,构建GFP(绿色荧光蛋白)融合蛋白,观察其亚细胞定位,利用酵母验证其转录激活活性,通过qRT-PCR分析在镉胁迫下该基因的表达特性和组织特异性。【结果】获得了1条新的东南景天WRKY转录因子家族基因并命名为SaWRKY7,包含完整的开放阅读框,长度为1 011 bp,共编码336个氨基酸,为WRKY基因家族Ⅱd成员。SaWRKY7定位在细胞核,属于核蛋白,不具有转录自激活活性。SaWRKY7在东南景天的根、茎、叶中均有表达,且在根中相对有较高的表达量。与对照相比,在100 μmol/L CdCl2处理0.5 h时,SaWRKY7在根茎叶中有较高的表达量,随后逐渐降低,属于早期诱导表达基因。【结论】SaWRKY7为WRKY转录因子家族成员,该基因定位在细胞核,不具有转录自激活活性,在早期响应镉胁迫诱导表达,推测其可能在东南景天镉积累或镉耐受过程中发挥重要作用。
Abstract:
【Objective】 Cadmium is highly toxic to plants. WRKY transcription factors are widely involved in stress response, therefor we studied the stress response and resistance mechanism of cadmium induced WRKY gene.【Method】 Gene sequences were obtained by PCR from the cDNA library of Sedum alfredii. Bioinformatics was used to analyze the protein structure.The green fluorescent protein fusion protein was constructed for the subcellular localization analysis. The transcriptional activation activity was verified with yeast. The expression characteristics and tissue specificity of the gene were analyzed by qRT-PCR under cadmium stress. 【Result】 A new member of the family of WRKY transcription factor genes was obtained and named SaWRKY7. It contained a complete open reading frame, which was 1 011 base pairs in length, encoded 336 amino acids, and was a member of the Ⅱd family of WRKY genes. SaWRKY7 was found in the nucleus, is a nucleoprotein, and showed no transcriptional activation activity. SaWRKY7 was expressed in roots, stems and leaves of S. alfredii, with relatively high expression found in roots. SaWRKY7 showed a higher expression level in the rhizomes and leaves in the 0.5 h with 100 μmol/L cadmium treatment than that of the control, and then decreased gradually; thus, it belonged to the early induced expression gene. 【Conclusion】 SaWRKY7 is a member of the family of WRKY transcription factor genes, which is found in the nucleus and does not have transcriptional self-activation activity. SaWRKY7 gene expression was induced by cadmium stress during the early stages. Results showed that SaWRKY7 might play an important role in S. alfredii response to cadmium stress.

参考文献/References:

[1] 环境保护部, 国土资源部. 全国土壤污染状况调查公报[J]. 中国环保产业, 2014, 36(5): 1689-1692.
Ministry of Environmental Protection, Ministry of Land and Resources. Bulletin of national soil pollution survey [J]. China Environmental Protection Industry, 2014, 36(5): 1689-1692.
[2] 庞荣丽, 王瑞萍, 谢汉忠, 等. 农业土壤中镉污染现状及污染途径分析[J]. 天津农业科学, 2016, 22(12): 87-91. DOI:10.3969/j.issn.1006-6500.2016.12.023.
PANG R L, WANG R P, XIE H Z, et al. Analysis of cadmium pollution in agricultural soils and analysis of its way of pollution[J]. Tianjin Agricultural Sciences, 2016, 22(12): 87-91.
[3] GALLEGO S M, PENA L B, BARCIA R A, et al. Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms[J]. Environmentaland Experimental Botany, 2012, 83: 33-46. DOI:10.1016/j.envexpbot.2012.04.006.
[4] 魏树和, 周启星, 王新, 等. 超积累植物龙葵及其对镉的富集特征[J]. 环境科学, 2005, 26(3): 167-171. DOI:10.3321/j.issn:0250-3301.2005.03.034.
WEI S H, ZHOU Q X, WANG X, et al. Cadmium-hyperaccumulator Solanum nigrum L. and its accumulating characteristics[J]. Chinese Journal of Environmental Science, 2005, 26(3): 167-171.
[5] 刘周莉, 何兴元, 陈玮. 忍冬: 一种新发现的镉超富集植物[J]. 生态环境学报, 2013, 22(4): 666-670. DOI:10.16258/j.cnki.1674-5906.2013.04.025.
LIU Z L, HE X Y, CHEN W. Lonicera japonica Thunb.: a newly discovered Cd hyper-accumulator[J]. Ecology and Environmental Sciences, 2013, 22(4): 666-670.
[6] 刘威, 束文圣, 蓝崇钰. 宝山堇菜(Viola baoshanensis): 一种新的镉超富集植物[J]. 科学通报, 2003, 48(19): 2046-2049. DOI:10.3321/j.issn:0023-074X.2003.19.009.
LIU W, SHU W S, LAN C Y. Viola baoshanensis: a new Cd hyper-accumulator [J]. Chinese Science Bulletin, 2003, 48(19): 2046-2049.
[7] KÜPPER H, LOMBI E, ZHAO F J, et al. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri[J]. Planta, 2000, 212(1): 75-84. DOI:10.1007/s004250000366.
[8] 韦朝阳, 陈同斌. 重金属污染植物修复技术的研究与应用现状[J]. 地球科学进展, 2002, 17(6): 833-839. DOI:10.3321/j.issn:1001-8166.2002.06.006.
WEI Z Y, CHEN T B. An pverview on the status of research and application of heavy metal phytormediation[J]. Advance in Earth Sciences, 2002, 17(6): 833-839.
[9] 安婧, 宫晓双, 魏树和. 重金属污染土壤超积累植物修复关键技术的发展[J]. 生态学杂志, 2015, 34(11): 3261-3270. DOI:10.13292/j.1000-4890.20151023.025.
AN J, GONG X S, WEI S H. Research progress on technologies of phytoremediation of heavy metal contaminated soils[J]. Chinese Journal of Ecology, 2015, 34(11): 3261-3270.
[10] LUO Z B, HE J L, POLLE A, et al. Heavy metal accumulation and signal transduction in herbaceous and woody plants: paving the way for enhancing phytoremediation efficiency[J]. Biotechnology Advances, 2016, 34(6): 1131-1148. DOI:10.1016/j.biotechadv.2016.07.003.
[11] OONO Y, YAZAWA T, KAWAHARA Y, et al. Genome-wide transcriptome analysis reveals that cadmium stress signaling controls the expression of genes in drought stress signal pathways in rice[J]. PLoS One, 2014, 9(5): e96946. DOI:10.1371/journal.pone.0096946.
[12] YUAN J B, BAI Y Q, CHAO Y H, et al. Genome-wide analysis reveals four key transcription factors associated with cadmium stress in creeping bentgrass(Agrostis stolonifera L.)[J]. Peer J, 2018, 6: e5191. DOI:10.7717/peerj.5191.
[13] FARINATI S, DALCORSO G, VAROTTO S, et al. The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants[J]. The New Phytologist, 2010, 185(4): 964-978. DOI:10.1111/j.1469-8137.2009.03132.x.
[14] TANG W, CHARLES T M, NEWTON R J. Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine(Pinus virginiana Mill.)confers multiple stress tolerance and enhances organ growth[J]. Plant Molecular Biology, 2005, 59(4): 603-617. DOI:10.1007/s11103-005-0451-z.
[15] ÜLKER B, SOMSSICH I E. WRKY transcription factors: from DNA binding towards biological function[J]. Current Opinion in Plant Biology, 2004, 7(5): 491-498. DOI:10.1016/j.pbi.2004.07.012.
[16] CHEN L G, SONG Y, LI S J, et al. The role of WRKY transcription factors in plant abiotic stresses[J]. Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms, 2012, 1819(2): 120-128. DOI:10.1016/j.bbagrm.2011.09.002.
[17] SONG Y, JING S J, YU D Q. Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis[J]. Chinese Science Bulletin, 2009, 54(24): 4671-4678. DOI:10.1007/s11434-009-0710-5.
[18] LEE H, CHA J, CHOI C, et al. Rice WRKY11 plays a role in pathogen defense and drought tolerance[J]. Rice, 2018, 11(1): 1-12. DOI:10.1186/s12284-018-0199-0.
[19] YOKOTANI N, SATO Y, TANABE S, et al. WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance[J]. Journal of Experimental Botany, 2013, 64(16): 5085-5097. DOI:10.1093/jxb/ert298.
[20] GU L, WEI H, WANG H, et al. Characterization and functional analysis of GhWRKY42, a group IId WRKY gene, in upland cotton(Gossypium hirsutum L.)[J]. Bmc Genetics, 2018, 19(1): 48. DOI: 10.1186/s12863-018-0653-4.
[21] ZHANG L L, CHENG J, SUN X M, et al. Overexpression of VaWRKY14 increases drought tolerance in Arabidopsis by modulating the expression of stress-related genes[J]. Plant Cell Reports, 2018. DOI: 10.1007/s00299-018-2302-9.
[22] MZID R, ZORRIG W, BEN AYED R, et al. The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum[J]. Biotech, 2018, 8(6): 1-15. DOI:10.1007/s13205-018-1301-4.
[23] HE L, WU Y H, ZHAO Q, et al. Chrysanthemum DgWRKY2 gene enhances tolerance to salt stress in transgenic chrysanthemum[J]. International Journal of Molecular Sciences, 2018, 19(7): E2062. DOI:10.3390/ijms19072062.
[24] VANDERAUWERA S, VANDENBROUCKE K, INZE A, et al. AtWRKY15 perturbation abolishes the mitochondrial stress response that steers osmotic stress tolerance in Arabidopsis[J]. Proceedings of the National Academy of Sciences, 2012, 109(49): 20113-20118. DOI:10.1073/pnas.1217516109.
[25] ALI M A, AZEEM F, NAWAZ M A, et al. Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis[J]. Journal of Plant Physiology, 2018, 226: 12-21. DOI:10.1016/j.jplph.2018.04.007.
[26] 彭喜旭, 白宁宁, 王海华. 响应镉胁迫的水稻WRKY15转录因子基因的分离与表达特征[J]. 中国水稻科学, 2018, 32(2): 103-110. DOI: 10.16819/j.1001-7216.2018.7056.
PENG X X, BAI N N, WANG H H. Isolation and expression profiles of cadmium stress-responsive rice WRKY15 transcription factor gene[J]. Chinese Journal of Rice Science, 2018, 32(2): 103-110.
[27] LIU Z Q, FANG H H, PEI Y X, et al. WRKY transcription factors down-regulate the expression of H2S-generating genes, LCD and DES in Arabidopsis thaliana[J]. Science Bulletin, 2015, 60(11): 995-1001. DOI:10.1007/s11434-015-0787-y.
[28] 汪祖昊. 地理隔离与重金属污染对东南景天物种分化的影响[D]. 广州: 中山大学, 2009.
WANG Z H. The effects of geographic isolation and heavy metal contamination on the speciation of Sedum alfredii[D]. Guangzhou: Sun Yat-sen University, 2009.
[29] 吴龙华, 周守标, 毕德, 等. 中国景天科植物一新种: 伴矿景天[J]. 土壤, 2006, 38(5): 632-633. DOI:10.3321/j.issn:0253-9829.2006.05.022.
WU L H, ZHOU S B, BI D, et al. Sedum plumbizincicola, a new species of the crassulaceae from Zhejiang, China[J]. Soils, 2006, 38(5): 632-633.
[30] WU L H, LIU Y J, ZHOU S B, et al. Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. wu(crassulaceae): a new species from Zhejiang Province, China[J]. Plant Systematics and Evolution, 2013, 299(3): 487-498. DOI:10.1007/s00606-012-0738-x.
[31] YANG X E, LONG X X, YE H B, et al. Cadmium tolerance and hyperaccumulation in a new Zn-hyperaccumulating plant species(Sedum alfredii Hance)[J]. Plant and Soil, 2004, 259(1/2): 181-189. DOI:10.1023/b:plso.0000020956.24027.f2.
[32] ZHANG M, ZHANG J, LU L L, et al. Functional analysis of CAX2-like transporters isolated from two ecotypes of Sedum alfredii[J]. Biologia Plantarum, 2016, 60(1): 37-47. DOI:10.1007/s10535-015-0557-3.
[33] ZHANG J, ZHANG M, TIAN S K, et al. Metallothionein 2(SaMT2)from Sedum alfredii Hance confers increased Cd tolerance and accumulation in yeast and tobacco[J]. PLoS One, 2014, 9(7): e102750. DOI:10.1371/journal.pone.0102750.
[34] LIU H, ZHAO H, WU L, et al. Heavy metal ATPase 3(HMA3)confers cadmium hypertolerance on the cadmium/zinc hyperaccumulator Sedum plumbizincicola[J]. New Phytologist, 2017, 215(2): 687-698. DOI: 10.1111/nph.14622.
[35] ZHANG M, SENOURA T, YANG X E, et al. Functional analysis of metal tolerance proteins isolated from Zn/Cd-hyperaccumulating ecotype and non-hyperaccumulating ecotype of Sedum alfredii Hance[J]. FEBS Letters, 2011, 585(16): 2604-2609. DOI:10.1016/j.febslet.2011.07.013.
[36] LIU M Y, QIU W M, HE X L, et al. Functional characterization of a gene in Sedum alfredii Hance resembling rubber elongation factor endowed with functions associated with cadmium tolerance[J]. Frontiers in Plant Science, 2016, 7: 965. DOI:10.3389/fpls.2016.00965.
[37] LI Z, HAN X J, SONG X X, et al. Overexpressing the Sedum alfredii Cu/Zn superoxide dismutase increased resistance to oxidative stress in transgenic Arabidopsis[J]. Frontiers in Plant Science, 2017, 8: 1010. DOI:10.3389/fpls.2017.01010.
[38] 赵婷. 东南景天耐镉相关基因SaLRR的克隆与功能初步分析[D]. 乌鲁木齐: 新疆大学, 2014.
ZHAO T. Cloning and molecular characterization of SaLRR gene in Sedum alfredii Hance[D]. Wulumuqi: Xinjiang University, 2014.
[39] CHEN S S, HAN X J, FANG J, et al. Sedum alfredii SaNramp6 metal transporter contributes to cadmium accumulation in transgenic Arabidopsis thaliana[J]. Scientific Reports, 2017, 7: 13318. DOI:10.1038/s41598-017-13463-4.
[40] ZHANG J, ZHANG M, SHOHAG M J I, et al. Enhanced expression of SaHMA3 plays critical roles in Cd hyperaccumulation and hypertolerance in Cd hyperaccumulator Sedum alfredii Hance[J]. Planta, 2016, 243(3): 577-589. DOI:10.1007/s00425-015-2429-7.
[41] 刘明英, 乔桂荣, 蒋晶, 等. 矿山型东南景天cDNA表达文库构建与耐镉基因筛选[J]. 林业科学研究, 2012, 25(3): 332-338. DOI:10.3969/j.issn.1001-1498.2012.03.010.
LIU M Y, QIAO G R, JIANG J, et al. Construction of stress induced full length cDNA library of Sedum alfredii and isolation of genes related to cd-tolerance[J]. Forest Research, 2012, 25(3): 332-338.
[42] HAN X, YIN H, SONG X. Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation[J]. Plant Biotechnology Journal, 2016, 14(6):1470-1483. DOI: 10.1111/pbi.12512.
[43] YOO S D, CHO Y H, SHEEN J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nature Protocols, 2007, 2(7): 1565-1572. DOI: 10.1038/nprot.2007.199.
[44] SANG J, HAN X J, LIU M Y, et al. Selection and validation of reference genes for real-time quantitative PCR in hyperaccumula-ting ecotype of Sedum alfredii under different heavy metals stresses[J]. PLoS One, 2013, 8(12): 82927. DOI:10.1371/journal.pone.0082927.
[45] EULGEM T, RUSHTON P J, ROBATZEK S, et al. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 2000, 5(5): 199-206. DOI:10.1016/s1360-1385(00)01600-9.
[46] PARK C Y, LEE J H, YOO J H, et al. WRKY group IId transcription factors interact with calmodulin[J]. FEBS Letters, 2005, 579(6): 1545-1550. DOI:10.1016/j.febslet.2005.01.057.
[47] LU L L, TIAN S K, ZHANG M, et al. The role of Ca pathway in Cd uptake and translocation by the hyperaccumulator Sedum alfredii[J]. Journal of Hazardous Materials, 2010, 183(1/2/3): 22-28. DOI:10.1016/j.jhazmat.2010.06.036.
[48] 廖星程. 东南景天对镉的细胞吸收与积累特征及其与钙的关系[D]. 杭州: 浙江大学, 2015.
LIAO X C. Cadium uptake and accumulation in Sedum alfredii at cellular levels and its association with calcium pathyway[D]. Hangzhou: Zhejiang University, 2015.
[49] SUN J, AN H, SHI W, et al. Molecular cloning and characterization of GhWRKY11, a gene implicated in pathogen responses from cotton[J]. South African Journal of Botany, 2012, 81: 113-123. DOI:10.1016/j.sajb.2012.06.005.
[50] 向小华, 吴新儒, 晁江涛, 等. 普通烟草WRKY基因家族的鉴定及表达分析[J]. 遗传, 2016, 38(9): 840-862. DOI:10.16288/j.yczz.16-016.
XIANG X H, WU X R, CHAO J T, et al. Genome-wide identification and expression analysis of the WRKY gene family in common tobacco(Nicotiana tabacum L.)[J]. Hereditas, 2016, 38(9): 840-862.
[51] XU Z L, RAZA Q, XU L, et al. GmWRKY49, a salt-responsive nuclear protein, improved root length and governed better salinity tolerance in transgenic Arabidopsis[J]. Frontiers in Plant Science, 2018, 9: 809. DOI:10.3389/fpls.2018.00809.
[52] 倪志勇, 加得拉·吐留汗, 邱迎风,等. 海岛棉GbWRKY40基因的克隆及特征分析[J]. 棉花学报, 2017, 29(4): 393-400. DOI: 10.11963/1002-7807. nzycqj.20170601.
NI Z Y, GARDELA T, QIU Y F, et al. Cloning and characterization of the GbWRKY40 transcription factor gene from Gossypium barbadense L.[J]. Cotton Science, 2017, 29(4): 393-400.
[53] ZHOU Q Y, TIAN A G, ZOU H F, et al. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J]. Plant Biotechnology Journal, 2008, 6(5): 486-503. DOI:10.1111/j.1467-7652.2008.00336.x.
[54] MUHAMMAD A J. CmWRKY4, CmWRKY10 and CmWRKY11 contribute to drought tolerance in chrysanthemum[D]. Nanjing: Nanjing Agricultural University, 2016.
[55] 蔡荣号. 玉米WRKY转录因子IId亚族抗逆相关基因的鉴定及ZmWRKY17的功能分析[D]. 合肥:安徽农业大学, 2016.
CAI R H. Identification of stress-resistant related genes of group IId WRKY transcription factor family in maize and function analysis of ZmWRKY17[D]. Hefei:Anhui Agricultural University, 2016.
[56] 司爱君, 余渝, 陈红,等. 棉花逆境应答GhWRKY2基因的结构与功能预测[J]. 农业生物技术学报, 2017, 25(2):222-230. DOI:10.3969/j.issn. 1674-7968.2017.02.006.
SI A J, YU Y, CHEN H, et al. Functional prediction of stress response GhWRKY2 gene in cotton(Gossypium hirsutum)[J].Journal of Agricultural Biotechnology, 2017, 25(2):222-230.
[57] 王玲,刘峰,戴明剑,等. 甘蔗ScWRKY4基因的克隆与表达特性分析[J]. 作物学报, 2018, 44(9): 1367-1379.
WANG L, LIU F, DAI M J, et al. Cloning and expression characteristic analysis of ScWRKY4 gene in sugarcane[J]. Acta Agronomica Sinica, 2018, 44(9): 1367-1379.
[58] BAO W, WANG X, CHEN M, et al. A WRKY transcription factor, PcWRKY33, from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana[J]. Plant Cell Reports, 2018, 37(7): 1033-1048. DOI: 10.1007/s00299-018-2289-2.

备注/Memo

备注/Memo:
收稿日期:2018-10-09 修回日期:2018-12-24
基金项目:国家重点研发计划子课题(2016YFD080080104)。
第一作者:王影(1120637812@qq.com)。*通信作者:曲同宝(qvtb@sina.com),副教授,ORCID(0000-0002-3777-120X),负责论文框架设定与修改; 卓仁英(zhuory@gmail.com),研究员,博士,ORCID(0000-0002-7063-3714),负责指导项目的具体实施。
更新日期/Last Update: 2019-05-15