Isolation and Characterization of a Novel Root Tipspecific Promoter in Rice

doi:10.3969/j.issn.1001-7216.2014.04.003

Abstract

Abstract: To obtain the rootspecific promoters in rice, a rootspecific gene Os04g24469 was selected by analyzing the public microarray data and further confirmed by RTPCR. To determine the expression pattern of the novel promoter, the putative promoter was isolated and fused with the GUS reporter gene to transform into the rice variety Nipponbare. Subsequent GUS staining of the transgenic plants showed that the Os04g24469 promoter was only activated in rice root tip. Using this root tipspecific promoter，we characterized the function of a ubiquitously expressed gene OsSRG in root development. The absence of OsSRG led to a short root phenotype in the srg mutant, the root tipspecific expression of GUSOsSRG in srg plants significantly reversed the root growth defect, indicating that the expression of OsSRG in root tip, not in shoot, is important for root development in rice. The present study showed that Os04g24469 promoter is highly active in the root tip and can be useful for the root tipspecific expression of target genes and gene function analysis in rice root.

Key words: rice, promoter, root tipspecific promoter, GUS reporter gene, SRG gene

摘要： 为了挖掘水稻中的根系特异表达启动子，通过生物信息学分析和RTPCR验证获得1个在根尖特异表达的水稻基因Os04g24469。用分离出的该基因上游启动子，与GUS 报告基因构建表达载体并转化到受体水稻日本晴中。通过对转基因后代中不同组织器官中的GUS活性检测，证明该启动子在水稻根尖特异性强烈表达。结合该根尖特异表达启动子，我们分析了一个在水稻地上部及地下部都组成表达的功能基因OsSRG对水稻根系发育的贡献。该基因的缺失导致水稻突变体srg根系变短，而利用分离的根尖特异性Os04g24469启动子在根尖驱动GUSOsSRG的表达，能回复突变体根系生长，表明OsSRG在根尖的表达足以影响水稻根系生长，而在地上部的表达与根系发育无关。本研究表明Os04g24469启动子是一个水稻根尖特异表达启动子，利用该启动子可以控制目标基因在水稻根尖特异表达，可用于水稻根系基因功能的研究。

关键词: 水稻, 启动子, 根尖特异表达启动子, GUS报告基因, SRG基因

CLC Number:

ZHAO Hongyu1, 2, XU Lei2, WEI Xijuan1, DENG Minjuan2, WANG Fang2,*, YI Keke2. Isolation and Characterization of a Novel Root Tipspecific Promoter in Rice[J]. Chinese Journal of Rice Science, 2014, 28(4): 351-357.

赵红玉1,2,徐磊2,魏溪涓1,邓敏娟2,王芳2,*,易可可2. 一个新水稻根尖特异表达启动子的分离与鉴定[J]. 中国水稻科学, 2014, 28(4): 351-357.

References

\[1\]Odell J T, Nagy F, Chua N H. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature, 1985, 313(28): 810812.

\[2\]Kim S R, Kim Y, An G. Identification of methyl jasmonate and salicylic acid response elements from the nopaline synthase (nos) promoter. Plant Physiol, 1993, 103(1): 97103.

\[3\]Christensen A H, Quail P H. Ubiquitin promoterbased vectors for highlevel expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res, 1996, 5(3): 213218.

\[4\]Christensen A H, Sharrock R A, Quail P H. Maize polyubiquitin genes: Structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol, 1992, 18(4): 675689.

\[5\]McElroy D, Blowers A D, Jenes B, et al. Construction of expression vectors based on the rice actin1 (Act1) 5′ region for use in monocot transformation. Mol Gen Genet, 1991, 231(1): 150160.

\[6\]Jang I C, Choi W B, Lee K H, et al. Highlevel and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots. Plant Physiol, 2002, 129(4): 14731481.

\[7\]Jeon J S, Lee S, Jung K H, et al. Tissuepreferential expression of a rice alphatubulin gene, OsTubA1, mediated by the first intron. Plant Physiol, 2000, 123(3): 10051014.

\[8\]Wang J, Oard J H. Rice ubiquitin promoters: Deletion analysis and potential usefulness in plant transformation systems. Plant Cell Rep, 2003, 22(2): 129134.

\[9\]Hsieh T H, Lee J T, Charng Y Y, et a1. Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol, 2002, 130(2): 618626.

\[10\]Pino M T, Skinner J S, Park E J, et al. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnol J, 2007, 5(5): 591604.

\[11\]Potenza C, Aleman L, SenguptaGopalan C. Targeting transgene expression in research, agricultural, and environmental applications: Promoters used in plant transformation. In Vitro Cell Dev Biol, 2004, 40(1): 122.

\[12\]郝中娜, 王海华, 郭泽建. 水稻OsWRKY89基因启动子的表达特性. 中国水稻科学, 2006, 20(2): 125130.

\[13\]Taniguchi M, Izawa K, Ku M S, et a1. The promoter for the maize C4 pyruvate, orthophosphate dikinase gene directs celland tissuespecific transcription in transgenic maize plants. Plant Cell Physiol, 2000, 41(1): 4248.

\[14\]李钱峰, 张桂云, 于恒秀, 等. 水稻异淀粉酶基因ISA1及其启动子的表达特性分析. 中国水稻科学, 2009, 23(1): 1218.

\[15\]马沁沁, 张金辉, 陈荣军, 等. 水稻花药绒毡层特异表达启动子的分离与表达载体构建. 应用环境生物学报, 2005, 11(4): 399403.

\[16\]武丽敏, 陈维, 赵艳, 等. 乔松根特异启动子PmPgPR10驱动Na+/H+逆转运蛋白提高转基因水稻的耐盐性. 中国水稻科学, 2012, 26(6): 643650.

\[17\]房孝良, 刘炜, 安静, 等. 水稻种胚特异性启动子OsESP1的克隆及其表达特性. 作物学报, 2011, 37(10): 19041909.

\[18\]Conkling M A, Cheng C L, Yamamoto Y T, et al. Isolation of transcriptionally regulated rootspecific genes from tobacco. Plant Physiol, 1990, 93(3): 12031211.

\[19\]Werner T, Nehnevajova E, Kllmer I, et al. Rootspecific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. Plant Cell, 2010, 22(12): 39053920.

\[20\]Jeong J S, Kim Y S, Baek K H, et al. Rootspecific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions. Plant Physiol, 2010, 153(1): 185197.

\[21\]Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res, 1980, 8(19): 43214325.

\[22\]Chen S Y, Jin W Z, Wang M Y, et al. Distribution and characterization of over 1000 TDNA tags in rice genome. Plant J, 2003, 36(1): 105113.

\[23\]Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: Betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6(13): 39013907.

\[24\]Hruz T, Laule O, Szabo G, et al. Genevestigator V3: A reference expression database for the metaanalysis of transcriptomes. Adv Bioinform, 2008, 2008: 420747420751.

\[25\]Wang L, Xie W, Chen Y, et al. A dynamic gene expression atlas covering the entire life cycle of rice. Plant J, 2010, 61(5): 752766.

\[26\]Li Y, Liu S, Yu Z, et al. Isolation and characterization of two novel rootspecific promoters in rice (Oryza sativa L.). Plant Sci, 2013, 207: 3744.

\[27\]Higo K, Ugawa Y, Iwamoto M, et al. Plant cisacting regulatory DNA elements (PLACE) database. Nucleic Acids Res, 1999, 27(1): 297300.

\[28\]Gowik U, Burscheidt J, Akyildiz M, et al. cisRegulatory elements for mesophyllspecific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene. Plant Cell, 2004, 16(5): 10771090.

\[29\]Rogers H J, Bate N, Combe J, et al. Functional analysis of cisregulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol, 2001, 45(5): 577585.

\[30\]Elmayan T, Tepfer M. Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Res, 1995, 4(6): 388396.

\[31\]Keller B, Baumgartner C. Vascularspecific expression of the bean GRP 1.8 gene is negatively regulated. Plant Cell, 1991, 3(10): 16391646.

\[32\]Jiang H W, Wang S M, Dang L, et al. A novel shortroot gene encodes a glucosamine6phosphate acetyltransferase required for maintaining normal root cell shape in rice. Plant Physiol, 2005, 138(1): 232242.

\[33\]Li J, Zhu S H, Song X W, et al. A rice glutamate receptorlike gene is critical for the division and survival of individual cells in the root apical meristem. Plant Cell, 2006, 18(2): 340349.

\[34\]Zhang J W, Xu L, Wu Y R, et al. OsGLU3, a putative membranebound endo1,4betaglucanase, is required for root cell elongation and division in rice (Oryza sativa L.). Mol Plant, 2012, 5(1): 176186.

[1]	REN Zhiqi, XUE Kexin, DONG Zheng, LI Xiaoxiang, LI Yongzhao, GUO Yujing, LIU Wenqiang, GUO Liang, SHENG Xinnian, LIU Zhixi, PAN Xiaowu. Identification and Gene Mapping of Outcurved Leaf Mutant ocl1 in Rice [J]. Chinese Journal OF Rice Science, 2023, 37(4): 337-346.
[2]	XIAO Lequan, LI Lei, DAI Weimin, QIANG Sheng, SONG Xiaoling. Seedling Growth Characteristics of Hybrids Between Transgenic Rice with cry2A/bar* Genes and Weedy Rice [J]. Chinese Journal OF Rice Science, 2023, 37(4): 347-358.
[3]	LI Gang, GAO Qingsong, LI Wei, ZHANG Wenxia, WANG Jian, CHEN Baoshan, WANG Di, GAO Hao, XU Weijun, CHEN Hongqi, JI Jianhui. Directed Knockout of SD1 Gene Improves Lodging Resistance and Blast Resistance of Rice [J]. Chinese Journal OF Rice Science, 2023, 37(4): 359-367.
[4]	WANG Shengyong, CHEN Yuhang, CHEN Huili, HUANG Yujie, ZHANG Xiaotian, DING Shuangcheng, WANG Hongwei. Effects of High Temperature on Phenylpropane Metabolism and Downstream Branch Metabolic Pathways in Rice Meiosis [J]. Chinese Journal OF Rice Science, 2023, 37(4): 368-378.
[5]	HUANG Yaru, XU Peng, WANG Lele, HE Yizhe, WANG Hui, KE Jian, HE Haibing, WU Liquan, YOU Cuicui. Effects of Exogenous Trehalose on Grain Filling Characteristics and Yield Formation of japonica Rice Cultivar W1844 [J]. Chinese Journal OF Rice Science, 2023, 37(4): 379-391.
[6]	DONG Liqiang, YANG Tiexin, LI Rui, SHANG Wenqi, MA Liang, LI Yuedong, SUI Guomin. Effect of Plant-row Spacing on Rice Yield and Root Morphological and Physiological Characteristics in Super High Yield Field [J]. Chinese Journal OF Rice Science, 2023, 37(4): 392-404.
[7]	GAO Qianqing, REN Xiaojian, ZHAI Zhongbing, ZHENG Pubing, WU Yuanfen, CUI Kehui. Effects of Panicle and Bud-promoting Nitrogen Fertilizer Application on Growth of Regenerated Bud and Grain Yield of Ratoon Rice [J]. Chinese Journal OF Rice Science, 2023, 37(4): 405-414.
[8]	HUANG Jinwen, LI Rikun, CHEN Zhicheng, ZHANG Bianhong, LEI Han, PAN Ruixin, YANG Mingyu, PAN Meiqing, TANG Lina. Effects of Straw Returning Techniques on Soil Nutrients, Organic Carbon and Microbial Diversity in Tobacco-rice Rotation System [J]. Chinese Journal OF Rice Science, 2023, 37(4): 415-426.
[9]	HAN Cong, HE Yuchang, WU Lijuan, JIA Lili, WANG Lei, E Zhiguo. Research Progress in the Function of Basic Leucine Zipper (bZIP) Protein Family in Rice [J]. Chinese Journal OF Rice Science, 2023, 37(4): 436-448.
[10]	SHEN Yumin, CHEN Mingliang, XIONG Huanjin, XIONG Wentao, WU Xiaoyan, XIAO Yeqing. Phenotypic Analysis and Fine Mapping of blg1(beak like grain 1), a Rice Mutant with Abnormal Palea and Lemma Development [J]. Chinese Journal OF Rice Science, 2023, 37(3): 225-232.
[11]	DUAN Min, XIE Liujie, GAO Xiuying, TANG Haijuan, HUANG Shanjun, PAN Xiaobiao. Creation of Thermo-sensitive Genic Male Sterile Rice Lines with Wide Compatibility Based on CRISPR/Cas9 Technology [J]. Chinese Journal OF Rice Science, 2023, 37(3): 233-243.
[12]	WANG Wenting, MA Jiaying, LI Guangyan, FU Weimeng, LI Hubo, LIN Jie, CHEN Tingting, FENG Baohua, TAO Longxing, FU Guanfu, QIN Yebo. Effect of Different Fertilizer Application Rates on Rice Yield and Quality Formation and Its Relationship with Energy Metabolism at High Temperature [J]. Chinese Journal OF Rice Science, 2023, 37(3): 253-264.
[13]	LIU Aihua, LI Xiaokun. Meta-analysis of Relationship Between Fertilizer Application and Rice Quality [J]. Chinese Journal OF Rice Science, 2023, 37(3): 276-284.
[14]	YANG Xiaolong, WANG Biao, WANG Benfu, ZHANG Zhisheng, ZHANG Zuolin, YANG Lantian, CHENG Jianping, LI Yang. Effects of Different Water Management on Yield and Rice Quality of Dry-seeded Rice [J]. Chinese Journal OF Rice Science, 2023, 37(3): 285-294.
[15]	WEI Xiaodong, SONG Xuemei, ZHAO Ling, ZHAO Qingyong, CHEN Tao, LU Kai, ZHU Zhen, HUANG Shengdong, WANG Cailin, ZHANG Yadong. Effects of Silicon and Zinc Fertilizer and Their Application Ways on Yield and Grain Quality of Rice Variety Nanjing 46 [J]. Chinese Journal OF Rice Science, 2023, 37(3): 295-306.