水稻转录因子OsWRKY68蛋白质的表达特征及其功能特性

doi:10.3864/j.issn.0578-1752.2019.12.001

摘要/Abstract

摘要：

【目的】水稻中有近百个WRKY转录因子家族成员,其中很多与生长发育、生物与非生物逆境胁迫应答有关。河北农业大学生命科学学院分子生物学与生物信息学实验室（molecular biology & bioinformatics lab,MBB）前期发现OsWRKY68在水稻接种白叶枯病菌后诱导表达,本研究试图进一步探究OsWRKY68的功能。【方法】采集水稻TP309不同生长发育时期的组织样品,包括萌发期、幼苗期、分蘖期、孕穗期和开花期的根、茎、叶、叶鞘、叶枕、穗子、花药、颖壳和种子,以及非生物胁迫（4℃、44℃、48℃、淹、NaCl、PEG、恒光和恒暗）和激素处理（脱落酸、茉莉酸甲酯、水杨酸和乙烯利）的叶片,提取总蛋白质,用水稻OsWRKY68蛋白质特异抗体,通过免疫印迹（western blot,WB）技术系统调查OsWRKY68蛋白质在水稻正常发育过程中不同时期、不同组织、非生物胁迫及激素处理条件下的表达特征。构建RNAi载体,通过农杆菌介导的方法转化水稻TP309,对转基因植株进行PCR和WB鉴定,观察OsWRKY68 RNAi转基因植株的表型并测量其株高、分蘖数、穗长、小穗数和结实率等性状。【结果】调查OsWRKY68蛋白质的表达丰度,发现OsWRKY68蛋白质在水稻正常生长发育过程中基本呈组成型表达,且在大部分组织中表达丰度差异倍数不大,但在开花期的花药中OsWRKY68表达量高于成熟穗、穗轴和颖壳;在分蘖期和孕穗期的叶鞘中不表达,仅在开花期的叶鞘中表达;在孕穗期的幼穗中,随着幼穗长度的增加其表达丰度逐渐降低。调查水稻非生物胁迫和激素处理后OsWRKY68蛋白质的表达特征,发现盐胁迫后OsWRKY68蛋白质的表达丰度随时间延长持续下降,恒光处理后OsWRKY68蛋白质表达量持续上升,3 d时明显出现一条分子质量较大的条带（记作OsWRKY68 ⁺）,且表达量逐渐增加,茉莉酸甲酯（methyl jasmonate,MeJA）和乙烯利（ethephon,ET）处理后同样也呈现OsWRKY68 ⁺蛋白质丰度的增加。对转基因植株进行鉴定和自交繁殖,对T₃代的4个OsWRKY68 RNAi转基因株系（Y316、Y317、Y326和Y337）进行PCR和WB鉴定,均表现阳性。在转基因植株中,OsWRKY68蛋白质的表达量均低于野生型TP309。对转基因植株进行表型观察和性状测量,发现与野生型相比转基因植株的株高降低、分蘖数和结实率下降等。【结论】 OsWRKY68蛋白质在水稻正常生长发育中发挥作用,敲低OsWRKY68蛋白质的表达能影响水稻的正常生长。OsWRKY68蛋白质可能参与盐、光照、茉莉酸甲酯和乙烯介导的信号转导过程。

关键词: 水稻, WRKY转录因子, 表达特征, 免疫印迹, RNA干扰

Abstract:

【Objective】 There are nearly 100 WRKY transcription factor members in rice genome, many of them are involved in plant growth and development, biotic and abiotic stress responses. Molecular biology & bioinformatics lab identified that the expression of OsWRKY68 protein was induced after inoculation with Xanthomonas oryzae pv. oryzae (Xoo) in rice. The aim of this study is attempt to further explore the function of OsWRKY68. 【Method】Rice TP309 samples of different tissues at different developmental stages, including germination, seedling, tillering, booting and flowering stages of root, stem, leaf, sheath, cushion, panicle, anther, husk, seed, abiotic stress (4℃, 44℃, 48℃, submerge, NaCl, PEG, constant light, constant dark) and hormone treatments (abscisic acid, methyl jasmonate, salicylic acid, ethephon) were collected. Total protein were extracted and analyzed by Western blot (WB) systematically using OsWRKY68-specific antibody. The expression patterns of OsWRKY68 protein isolated from different tissues at different developmental stages, and tissues obtained from abiotic stresses and hormone treatments were investigated. RNA interfering vector was constructed and transformed to wildtype TP309 rice variety via Agrobacterium tumefaciens strategy. Identification of transgenic plants were carried out by PCR and WB. The phenotype of OsWRKY68 RNAi transgenic plants were monitored and plant height, tiller number, spike length, spikelet number and seed-setting rate were measured.【Result】By comparing the abundance of OsWRKY68 protein in different tissues, it was found that OsWRKY68 protein was expressed in a constitutive way during the normal growth and development of rice, the abundance of OsWRKY68 protein expressed among different tissues were not varied too much. However, different levels of OsWRKY68 were observed. The expression level of OsWRKY68 in anthers at flowering stage was higher than that in mature panicles, panicle axis and husk. It was not expressed in sheaths at tillering and booting stages, but it was expressed in sheaths at flowering stage. In panicles, the abundance of OsWRKY68 was decreased gradually along with the growth of the young panicle. By investigating the expression patterns of OsWRKY68 protein under abiotic stress and hormone treatments, it was found that the abundance of OsWRKY68 protein decreased steadily under salt stress. The expression of OsWRKY68 protein increased steadily at constant light treatment, a specific band (designated as OsWRKY68 ⁺) with higher molecular weight appeared at three days and enhanced in the following timepoints. After methyl jasmonate (MeJA) and ethephon (ET) treatments, OsWRKY68 ⁺ band appeared also and its intensity increased as the treatments continues. Four homozygous OsWRKY68 RNAi transgenic lines (Y316, Y317, Y326 and Y337) were checked by PCR and WB analyses and verified at T₃ generation. The abundance of OsWRKY68 protein in RNAi transgenic plants was lower than that in wildtype TP309. Phenotypic investigation revealed significant reduction in plant height, tiller number and seed setting rate in transgenic plants.【Conclusion】Rice OsWRKY68 protein plays an important role in the process of normal growth and development of rice. Knocking down the abundance of OsWRKY68 protein via RNAi affected the normal growth of rice. In addition, the data of expression patterns suggested that the function of OsWRKY68 protein may be involved with salt stress, light, MeJA and ethene-mediated signal transduction pathways.

Key words: rice, WRKY transcription factor, expression patterns, western blot, RNA interference

陈悦, 王田幸子, 杨烁, 张彤, 马金姣, 燕高伟, 刘玉晴, 周艳, 史佳楠, 兰金苹, 魏健, 窦世娟, 刘丽娟, 杨明, 李莉云, 刘国振. 水稻转录因子OsWRKY68蛋白质的表达特征及其功能特性[J]. 中国农业科学, 2019, 52(12): 2021-2032.

Yue CHEN, TianXingZi WANG, Shuo YANG, Tong ZHANG, JinJiao MA, GaoWei YAN, YuQing LIU, Yan ZHOU, JiaNan SHI, JinPing LAN, Jian WEI, ShiJuan DOU, LiJuan LIU, Ming YANG, LiYun LI, GuoZhen LIU. Expression Profiling and Functional Characterization of Rice Transcription Factor OsWRKY68[J]. Scientia Agricultura Sinica, 2019, 52(12): 2021-2032.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.12.001

https://www.chinaagrisci.com/CN/Y2019/V52/I12/2021

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 44

[1]	VIANA V E, BUSANELLO C , DA MAIA L C, PEGORARO C, COSTA DE OLIVEIRA A. Activation of rice WRKY transcription factors: an army of stress fighting soldiers? Current Opinion Plant Biology, 2018,45:268-275. doi: 10.1016/j.pbi.2018.07.007
[2]	ROSS C A, LIU Y, SHEN Q J . The WRKY gene family in rice ( Oryza sativa). Journal of Integrative Plant Biology, 2007,49(6):827-842.
[3]	Rice WRKY Working Group . Nomenclature report on rice WRKY's - Conflict regarding gene names and its solution. Rice (New York), 2012,5(1):3.
[4]	CAI Y H, CHEN X J, XIE K, XING Q K, WU Y W, LI J, DU C H, SUN Z X, GUO Z J . Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice. Plos One, 2014,9(7):e102529. doi: 10.1371/journal.pone.0102529
[5]	LEE H, CHA J, CHOI C, CHOI N, JI H S, PARK S R, LEE S, HWANG D J . Rice WRKY11 plays a role in pathogen defense and drought tolerance. Rice (New York), 2018,11(1):5.
[6]	QIU D Y, XIAO J, XIE W B, LIU H B, LI X H, XIONG L Z, WANG S P . Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance. Molecular Plant, 2008,1(3):538-551.
[7]	XIAO J, CHENG H T, LI X H, XIAO J H, XU C G, WANG S P . Rice WRKY13 regulates cross talk between abiotic and biotic stress signaling pathways by selective binding to different cis-elements. Plant Physiology, 2013,163(4):1868-1882. doi: 10.1104/pp.113.226019
[8]	QIU D Y, XIAO J, DING X H, XIONG M, CAI M, CAO Y L, LI X H, XU C G, WANG S P . OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate- dependent signaling. Molecular Plant-Microbe Interactions, 2007,20(5):492. doi: 10.1094/MPMI-20-5-0492
[9]	ABBRUSCATO P, NEPUSZ T, MIZZI L, CORVO M D, MORANDINI P, FUMASONI I, MICHEL C, PACCANARO A, GUIDERDONI E, SCHAFFRATH U . OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast. Molecular Plant Pathology, 2012,13(8):828-841. doi: 10.1111/mpp.2012.13.issue-8
[10]	CHUJO T, MIYAMOTO K, SHIMOGAWA T, SHIMIZU T, OTAKE Y, YOKOTANI N, NISHIZAWA Y, SHIBUYA N, NOJIRI H, YAMANE H . OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Molecular Biology, 2013,82(1/2):23-37. doi: 10.1007/s11103-013-0032-5
[11]	彭喜旭, 胡耀军, 唐新科, 周平兰, 邓小波, 王海华 . 茉莉酸和真菌病原诱导的水稻WRKY30转录因子基因的分离及表达特征. 中国农业科学, 2011,21(44):2454-2461.
	PENG X X, HU Y J, TANG X K, ZHOU P L, DENG X B, WANG H H . Isolation and expression profiles jasmonic acid application and of rice WRKY30 induced by fungal pathogen infection. Scientia Agricultura Sinica, 2011,21(44):2454-2461. (in Chinese)
[12]	SHEN H S, LIU C T, ZHANG Y, MENG X P, ZHOU X, CHU C C, WANG X P . OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice. Plant Molecular Biology, 2012,80(3):241-253. doi: 10.1007/s11103-012-9941-y
[13]	ZHANG J, PENG Y L, GUO Z J . Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants. Cell Research, 2008,18(4):508-521. doi: 10.1038/cr.2007.104
[14]	HAN M K C, LEE J, LEE S K, JEON J S . OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice. Molecules & Cells, 2014,37(7):532.
[15]	PILLAI S E, KUMAR C, PATEL H K, SONTI R V . Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection. BMC Plant Biology, 2018,18(1):177. doi: 10.1186/s12870-018-1391-5
[16]	SHIMONO M, KOGA H, AKAGI A, HAYASHI N, GOTO S, SAWADA M, KURIHARA T, MATSUSHITA A, SUGANO S, JIANG C J . Rice WRKY45 plays important roles in fungal and bacterial disease resistance. Molecular Plant Pathology, 2011,13(1):83-94.
[17]	SHIMONO M, SUGANO S, NAKAYAMA A, JIANG C J, ONO K, TOKI S, TAKATSUJI H . Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. The Plant Cell, 2007,19(6):2064-2076. doi: 10.1105/tpc.106.046250
[18]	CHENG H T, LIU H B, DENG Y K, XIAO J X, LI X H, WANG S P . The WRKY45-2-WRKY13-WRKY42 transcriptional regulatory cascade is required for rice resistance to fungal pathogen. Plant Physiology, 2015,167(3):1087-1099. doi: 10.1104/pp.114.256016
[19]	QIU Y P, YU D Q . Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. Environmental and Experimental Botany, 2009,65:35-47.
[20]	WANG J, ZHOU L, SHI H, CHERN M, YU H, YI H, HE M, YIN J J, ZHU X B, LI Y, LI W T, LIU J L, WANG J C, CHEN X Q, QING H, WANG Y P, LIU G F, WANG W M, LI P, WU X J, ZHU L H, ZHOU J M, RONALD P, LI S G, LI J Y, CHEN X W . A single transcription factor promotes both yield and immunity in rice. Science, 2018,361:1026-1028. doi: 10.1126/science.aat7675
[21]	WEI T, OU BIN, LI J B, ZHAO Y, GUO D S, ZHU Y Y, CHEN Z L, GU H Y, LI C Y, QIN G J . Transcriptional profiling of rice early response to Magnaporthe oryzae identified OsWRKYs as important regulators in rice blast resistance. PLoS ONE, 2013,8(3):e59720.
[22]	RAINERI J, WANG S H, PELEG Z, BLUMWALD E, CHAN R L . The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress. Plant Molecular Biology, 2015,88(4/5):401-413. doi: 10.1007/s11103-015-0329-7
[23]	TIAN X J, LI X F, ZHOU W J, REN Y K, WANG Z Y, LIU Z Q, TANG J Q, TONG H N, FANG J, BU Q Y . Transcription factor OsWRKY53 positively regulates brassinosteroid signaling and plant architecture. Plant Physiology, 2017,175(3):1337-1349. doi: 10.1104/pp.17.00946
[24]	CHUJO T, TAKAI R, AKIMOTO-TOMIYAMA C, ANDO S, MINAMI E, NAGAMURA Y, KAKU H, SHIBUYA N, YASUDA M, NAKASHITA H, UMEMURA K, OKADA A, OKADA K, NOJIRI H, YAMANE H . Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice. Biochimica et Biophysica Acta-Biomembranes, 2007,1769(7/8):497-505. doi: 10.1016/j.bbaexp.2007.04.006
[25]	PENG Y, BARTLEY L E, CHEN X W, DARDICK C, CHERN M, RUAN R, CANLAS P E, RONALD P C . OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Molecular Plant, 2008,1(3):446-458.
[26]	VO K T X, KIM C Y, HOANG T V, LEE S K, SHIRSEKAR G, SEO Y S, LEE S W, WANG G L, JEON J S . OsWRKY67 plays a positive role in basal and XA21-mediated resistance in rice. Frontiers in Plant Science, 2018,8:2220.
[27]	LIU X Q, BAI X Q, WANG X J, CHU C C . OsWRKY71, a rice transcription factor, is involved in rice defense response. Journal of Plant Physiology, 2007,164(8):969-979. doi: 10.1016/j.jplph.2006.07.006
[28]	SONG Y, CHEN L G, ZHANG L P, YU D Q . Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis. Journal of Biosciences, 2010,35(3):459-471.
[29]	DAI X Y, WANG Y Y, ZHANG W H . OsWRKY74, a WRKY transcription factor, modulates tolerance to phosphate starvation in rice. Journal of Experimental Botany, 2016,67(3):947-960. doi: 10.1093/jxb/erv515
[30]	SEO Y S, CHERN M, BARTLEY L E, HAN M, JUNG K H, LEE I, WALIA H, RICHTER T, XU X, CAO P J . Towards establishment of a rice stress response interactome. Plos Genetics, 2011,7(4):e1002020. doi: 10.1371/journal.pgen.1002020
[31]	LIU Q Q, ZHANG C Q, XU Y, LU Y, YU H X, GU M H . The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice. Planta, 2011,234(3):541-554. doi: 10.1007/s00425-011-1423-y
[32]	WANG H H, HAO J J, CHEN X J, HAO Z N, WANG X, LOU Y G, PENG Y L, GUO Z J . Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Molecular Biology, 2007,65(6):799-815. doi: 10.1007/s11103-007-9244-x
[33]	YANG S, ZHOU L, MIAO L Y, SHI J N, SUN C Q, FAN W, LAN J P, CHEN H, LIU L J, DOU S J, LIU G Z, LI L Y . The expression and binding properties of the rice WRKY68 protein in the Xa21-mediated resistance response to Xanthomonas oryzae pv.Oryzae. Journal of Integrative Agriculture, 2016,15(11):2451-2460.
[34]	NURUZZAMAN M S A, SATOH K, KUMAR A, LEUNG H, KIKUCHI S . Comparative transcriptome profiles of the WRKY gene family under control, hormone-treated, and drought conditions in near-isogenic rice lines reveal differential, tissue specific gene activation. Journal of Plant Physiology, 2014,171(1):2-13. doi: 10.1016/j.jplph.2013.09.010
[35]	SATOH K, SAJI S, ITO S, SHIMIZU H, SAJI H, KIKUCHI S . Gene response in rice plants treated with continuous fog influenced by pH, was similar to that treated with biotic stress. Rice, 2014,7(1):10. doi: 10.1186/s12284-014-0010-9
[36]	RAMAMOORTHY R, JIANG S Y, KUMAR N, VENKATESH P N, RAMACHANDRAN S . A comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments. Plant Cell Physiology, 2008,49(6):865-879. doi: 10.1093/pcp/pcn061
[37]	刘国振, 刘斯奇, 吴琳, 徐宁志 . 基于抗体的水稻蛋白质组学策略——开端与展望. 中国科学, 2011,41(3):173-177.
	LIU G Z, LIU S Q, WU L, XU N Z . Antibody-based rice proteomics- the beginning and perspectives. Scientia Sinica Viate, 2011,41(3):173-177. (in Chinese)
[38]	张剑硕, 马金姣, 张彤, 陈悦, 魏健, 张柳, 史佳楠, 徐珊, 燕高伟, 杜铁民, 窦世娟, 李莉云, 刘丽娟, 刘国振 . 水稻蛋白质样品资源库RiceS-A300的建立与应用. 中国农业科学, 2018,51(19):3625-3638.
	ZHANG J S, MA J J, ZHANG T, CHEN Y, WEI J, ZHANG L, SHI J N, XU S, YAN G W, DU T M, DOU S J, LI L Y, LIU L J, LIU G Z . The establishment and application of rice protein sample library RiceS-A300. Scientia Agricultura Sinica, 2018,51(19):3625-3638. (in Chinese)
[39]	LI X M, BAI H, WANG X Y, LI L Y, CAO Y H, WEI J, LIU Y M, LIU L J, GONG X D, WU L, LIU S Q, LIU G Z . Identification and validation of rice reference proteins for western blotting. Journal of Experimental Botany, 2011,62:4763-4772. doi: 10.1093/jxb/err084
[40]	BAI H, LAN J P, GAN Q, WANG X Y, HOU M M, CAO Y H, LI L Y, LIU L J, HAO Y J, YIN C C, WU L, ZHU L H, LIU G Z . Identification and expression analysis of components involved in rice Xa21-mediated disease resistance signaling. Plant Biology, 2012,14:914-922.
[41]	LANDI S, HAUSMAN J F, GUERRIERO G, ESPOSITO S . Poaceae vs. abiotic stress: Focus on drought and salt stress, recent insights and perspectives. Frontiers in Plant Science, 2017,8:1214. doi: 10.3389/fpls.2017.01214
[42]	TAO Z, KOU Y J, LIU H B, LI X H, XIAO J H, WANG S P . OsWRKY45 alleles play different roles in abscisic acid signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice. Journal of Experimental Botany, 2011,62(14):4863-4874. doi: 10.1093/jxb/err144
[43]	CHEN H, LAI Z B, SHI J W, XIAO Y, CHEN Z X, XU X P . Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress. BMC Plant Biology, 2010,10:281.
[44]	李莉云, 史佳楠, 杨烁, 孙财强, 刘国振 . 基于转录特征的水稻WRKY转录因子功能注释. 遗传, 2016,38(2):126-136.
	LI L Y, SHI J N, YANG S, SUN C Q, LIU G Z . Functional annotation of rice WRKY transcription factors based on their transcriptional features. Hereditas, 2016,38(2):126-136. (in Chinese)