欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (7): 1029-1037.doi: 10.3724/SP.J.1006.2019.83070

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米ZmGRAS31基因的克隆及功能研究

殷龙飞1,2,王朝阳1,吴忠义2,张中保2,*(),于荣1,*()   

  1. 1 首都师范大学生命科学学院, 北京 100048
    2 北京市农林科学院农业生物技术研究中心 / 北京市农业基因资源和生物技术重点实验室, 北京 100097
  • 收稿日期:2018-11-07 接受日期:2019-01-19 出版日期:2019-07-12 网络出版日期:2019-03-22
  • 通讯作者: 张中保,于荣
  • 作者简介:E-mail: 18810259769@163.com
  • 基金资助:
    本研究由北京市自然科学基金项目(6172007);国家自然科学基金项目(31871351);北京市农林科学院科技创新能力建设专项(KJCX20180404)

Cloning and functional analysis of ZmGRAS31 gene in maize

YIN Long-Fei1,2,WANG Zhao-Yang1,WU Zhong-Yi2,ZHANG Zhong-Bao2,*(),YU Rong1,*()   

  1. 1 College of Life Sciences, Capital Normal University, Beijing 100048
    2 Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2018-11-07 Accepted:2019-01-19 Published:2019-07-12 Published online:2019-03-22
  • Contact: Zhong-Bao ZHANG,Rong YU
  • Supported by:
    This study was supported by the Beijing Natural Science Foundation(6172007);the National Natural Science Foundation of China(31871351);the Beijing Academy of Agricultural and Forestry Sciences(KJCX20180404)

RichHTML

37

PDF

666

摘要：

GRAS蛋白家族是一类植物特有的转录调控因子, 在多种植物中均有报道。为了探究玉米GRAS基因家族在逆境胁迫下的功能, 本研究从玉米根中克隆获得ZmGRAS31 (AC: NC_024462), 该基因全长1422 bp, 编码473个氨基酸。生物信息学分析表明ZmGRAS31蛋白分子量为51,700.38 Da, 理论等电点为4.73, 具有GRAS转录因子家族特有的保守结构域, 但不具跨膜结构, 亲水性较差。玉米原生质体瞬时表达实验表明ZmGRAS31定位于细胞核内。实时荧光定量PCR (qPCR)分析表明, 在低温、脱水、高盐、干旱处理下, ZmGRAS31在玉米幼苗均上调表达; 不同浓度NaCl处理过表达ZmGRAS31转基因拟南芥植株, 其根长优于野生型, 由此推测该基因可能参与玉米非生物胁迫应答。

关键词: 玉米, ZmGRAS31, 转录因子, 原生质体, qPCR, 非生物胁迫

Abstract:

The GRAS gene family is a kind of plant-specific transcription factor reported in many plants. To figure out the function of maize GRAS family under stress, we obtained ZmGRAS319 (AC: NC_024462) gene from maize (Zea mays) root. Sequence analysis showed that the coding sequence (CDS) of ZmGRAS31 was 1422 bp encoding a protein of 473 amino acids with molecular weight of 51,700.38 Da and an isoelectric point of 4.73. Bioinformatics analysis revealed that ZmGRAS31 had a conserved domain unique to the GRAS transcription factor family and poor hydrophilicity, but no transmembrane structure. Transient expression of maize protoplasts indicated that ZmGRAS31 was localized in the nucleus. Real-time quantitative PCR analysis showed that the expression of ZmGRAS31 was up-regulated in maize seedlings under low temperature, dehydration, high salinity and drought conditions. The root length of the ZmGRAS31 overexpression transgenic Arabidopsis plants was longer than that of wide type under different concentrations of NaCl treatments. Therefore, our data suggested that ZmGRAS31 might be involved in abiotic stress response.

Key words: maize, ZmGRAS31, transcription factor, protoplast, qPCR, abiotic stress

表1

本实验中所用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
pZmGRAS31-F AAAGCCGATGGAGACCATTTCAT
pZmGRAS31-R CTTTCATACAGGAGTCCAAGCA
pZmGRAS31SL-F CATATCGAATTCATGGAGACCATTTCATATCC
pZmGRAS31SL-R CCTAGCGGTACCTACAGGAGTCCAAGCAGATAT
pZmGRAS31RT-F CTTCGACACCATTGCTATTGG
pZmGRAS31RT-R CTTGAATCTCCAGCCTCAAC
pGAPDHRT-F CCCTTCATCACCACGGACTAC
pGAPDHRT-R AACCTTCTTGGCACCACCCT
pZmGRAS31At-F CCTATCGGCGCCATGGAGACCATTTCATATCC
pZmGRAS31At-R CCTAGCACCGGTTCATACAGGAGTCCAAGCAG

图1

玉米ZmGRAS31基因保守结构域预测"

图2

玉米ZmGRAS31蛋白在原生质体中的亚细胞定位 GFP: GFP标记的ZmGRAS31基因在玉米原生质体的亚细胞定位; DAPI: DAPI标记的原生质体细胞核; Bright: 同一视野光镜下的原生质体; Merged: 光镜及荧光照片的叠加; CK: 转入空载体的原生质体; ZmGRAS31: 转入目的载体的原生质体。Bar=10 μm。"

图3

ZmGRAS31在玉米不同组织中的相对表达量"

图4

实时荧光定量PCR分析不同处理条件下ZmGRAS31基因在地上部和根中的表达情况 A: 脱水处理; B: 低温(4°C)处理; C: 高盐(200 mmol L-1 NaCl); D: 干旱处理。数据为3个生物学重复±标准差。"

图5

不同盐浓度下转基因拟南芥根长比较 A~D分别为0 mol L-1、0.10 mol L-1、0.15 mol L-1、0.20 mol L-1 NaCl处理的生长情况; E: 不同盐浓度下拟南芥平均主根长度; WT: 野生型拟南芥; OE: 过表达拟南芥。Bar = 1.5 cm。"

图6

不同甘露醇浓度下转基因拟南芥根长比较 A~D分别为0 mol L-1、0.15 mol L-1、0.30 mol L-1、0.45 mol L-1甘露醇处理的生长情况; E: 不同甘露醇浓度下拟南芥平均主根长度, WT: 野生型拟南芥; OE: 过表达拟南芥。Bar=1.5 cm。"

[1] 赫福霞, 李柱刚, 阎秀峰, 王秀君 . 渗透胁迫条件下玉米萌芽期抗旱性研究. 作物杂志, 2014, ( 5):144-147.
He F X, Li Z G, Yan X F, Wang X J . Effect of osmotic stress on drought resistance of maize at germination stage. Crops, 2014, ( 5):144-147 (in Chinese with English abstract).
[2] 余爱丽, 赵晋锋, 王高鸿, 杜艳伟, 李颜方, 张正 . 玉米ZmSAMS1基因在盐、干旱等逆境胁迫下的表达分析. 玉米科学, 2016,24(3):31-35.
Yu A L, Zhao J F, Wang G H, Du Y W, Li Y F, Zhang Z . Expression analysis of maize ZmSAMS1 gene under salt, drought and other Stresses. J Maize Sci, 2016,24(3):31-35 (in Chinese with English abstract).
[3] 决登伟, 桑雪莲, 舒波, 刘丽琴, 石胜友 . 玉米ZmWRKY53- like与ZmWRKY67-like基因在非生物胁迫下的表达. 贵州农业科学, 2017,45(6):15-20.
Jue D W, Sang X L, Shu B, Liu L Q, Shi S Y . Expression of maize ZmWAKY53-like and ZmWRKY67-like gene under abiotic stresses. Guizhou Agric Sci, 2017,45(6):15-20 (in Chinese with English abstract).
[4] Li S F, Parish R W . Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products. Plant J, 1995,8:963-972.
[5] Kwon Y, Kim J H, Nguyen H N, Jikumaru Y, Kamiya Y, Hong S W, Lee H . A novel Arabidopsis MYB-like transcription factor, MYBH, regulates hypocotyl elongation by enhancing auxin accumulation. J Exp Bot, 2013,64:3911-3922.
[6] Su L T, Li J W, Liu D Q, Zhai Y, Zhang H J, Li X W, Zhang Q L, Wang Y, Wang Q Y . A novel MYB transcription factor, GmMYBJ1, from soybean confers drought and cold tolerance in Arabidopsis thaliana. Gene, 2014,538:46-55.
[7] Oeda K, Salinas J, Chua N H . A tobacco bZip transcription activator (TAF-1) binds to a G-box-like motif conserved in plant genes. EMBO J, 1991,10:1793-1802.
doi: 10.1002/embj.1991.10.issue-7
[8] Finkelstein R R, Lynch T J . The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell, 2000,12:599-609.
[9] 曹红利, 岳川, 周艳华, 王璐, 郝心愿, 杨亚军, 王新超 . 茶树bZIP转录因子基因CsbZIP1的克隆与表达定位. 作物学报, 2014,40:1702-1709.
Cao H L, Yue C, Zhou Y H, Wang L, Hao X Y, Yang Y J, Wang X C . Molecular cloning and expression of a bZIP transcription factor gene CsbZIP1 in tea plant(Camellia sinensis). Acta Agron Sin, 2014,40:1702-1709 (in Chinese with English abstract).
[10] Ali N, Hadi F . CBF/DREB transcription factor genes play role in cadmium tolerance and phytoaccumulation in Ricinus communis under molybdenum treatments. Chemosphere, 2018,208:425-432.
[11] Jaglo-Ottosen K R, Gilmour S J, Zarka D G, Schabenberger O, Thomashow M F . Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 1998,280:104-106.
[12] Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K . Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low- temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998,10:1391-1406.
[13] Souer E, van Houwelingen A, Kloos D, Mol J, Koes R . The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell, 1996,85:159-170.
[14] Fang Y, You J, Xie K, Xie W, Xiong L . Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NAC transcription factor family in rice. Mol Genet Genomics, 2008,280:547-563.
doi: 10.1007/s00438-008-0386-6
[15] Rushton P J, Bokowiec M T, Han S, Zhang H, Brannock J F, Chen X, Laudeman T W, Timko M P . Tobacco transcription factors: novel insights into transcriptional regulation in the solanaceae. Plant Physiol, 2008,147:280-295.
doi: 10.1104/pp.107.114041
[16] Ishiguro S, Nakamura K . Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato. Mol Genet Genetics, 1994,244:563-571.
[17] Du L, Chen Z . Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis. Plant J, 2000,24:837-847.
[18] Schrick K, Nguyen D, Karlowski W M, Mayer K F . START lipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors. Genome Biol, 2004,5:R41.
doi: 10.1186/gb-2004-5-6-r41
[19] Pysh L D, Wysocka-Diller J W, Camilleri C, Bouchez D, Benfey P N . The GRAS gene family in Arabidopsis: Sequence characterization and basic expression analysis of the SCARECROW- LIKE genes. Plant J, 1999,18:111-119.
[20] Hirsch S, Oldroyd G E D . GRAS-domain transcription factors that regulate plant development. Plant Signal Behavior, 2009,4:698-700.
doi: 10.4161/psb.4.8.9176
[21] Stuurman J, Jäggi F, Kuhlemeier C . Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells. Gene Dev, 2002,16:2213-2218.
[22] Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, Hauser M T, Benfey P N . The SHORT-ROOT, gene controls radial patterning of the Arabidopsis root through radial signaling. Cell, 2000,101:555-567.
[23] Yoo S D, Cho Y H, Sheen J . Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols, 2007,2:1565-1572.
[24] Livak K J, Schmittgen T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆CT method . Methods, 2001,25:402-408.
doi: 10.1006/meth.2001.1262
[25] Clough S J, Bent A F . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998,16:735-743.
[26] Thornsberry J M, Goodman M M, Doebley J, Kresovich S, Nielsen D, Buckler IV E S . Dwarf8 polymorphisms associate with variation in flowering time. Nat Genet, 2001,28:286-289.
[27] Lawit S J, Wych H M, Xu D, Kundu S, Tomes D T . Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant development. Plant Cell Physiol, 2010,51:1854-1868.
doi: 10.1093/pcp/pcq153
[28] Lim J, Helariutta Y, Specht C D, Jung J, Sims L, Bruce W B, Diehn S, Benfey P N . Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems. Plant Cell, 2000,12:1307-1318.
[29] Tanabe S, Onodera H, Hara N, Ishii-Minami N, Day B, Fujisawa Y, Hagio T, Toki S, Shibuya N, Nishizawa Y, Minami E . The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23. Biosci Biotechnol Biochem, 2016,80:145-151.
[30] 李丽丽, 曾卫军, 李艳红, 葛风伟, 卢函, 雷维, 杜钰, 谢红桃, 赵和平, 赵惠新 . 独行菜GRAS转录因子家族分析及LaSCL18基因克隆与冷相关性研究. 分子植物育种, 2017,15:3428-3437.
Li L L, Zeng W J, Li Y H, Ge F W, Lu H, Lei W, Du Y, Xie H T, Zhao H P, Zhao H X . Analysis of GRAS family transcription factors, cloning and researching cold tolerance of LaSCL18 gene in Lepidium. Mol Plant Breed, 2017,15:3428-3437 (in Chinese with English abstract).
[31] 郭华军, 焦远年, 邸超, 姚冬霞, 张盖华, 郑雪, 刘岚, 张群莲, 郭蔼光, 苏震 . 拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索. 植物学报, 2009,44:290-299.
Guo H J, Jiao Y N, Di C, Yao D X, Zhang G H, Zheng X, Liu L, Zhang Q L, Guo A G, Su Z . Discovery of Arabidopsis GRAS family genes in response to osmotic and drought stresses. Chin Bull Bot, 2009,44:290-299 (in Chinese with English abstract).
[32] 马洪双, 夏新莉, 尹伟伦 . 胡杨SCL7基因及其启动子片段的克隆与分析. 北京林业大学学报, 2011,33(1):1-10.
Ma H S, Xia X L, Yin W L . Cloning and analysis of SCL7 gene from Populus euphratica. J Beijing For Univ, 2011,33(1):1-10 (in Chinese with English abstract).
[33] 张焕欣, 董春娟, 尚庆茂 . 辣椒GRAS家族全基因组鉴定与表达分析. 园艺学报, 2017,44:2305-2317.
Zhang H X, Dong C J, Shang Q M . Genome-wide identification and expression analysis of GRAS gene family in Pepper. Acta Hortic Sin, 2017,44:2305-2317 (in Chinese with English abstract).
[34] 郭鹏, 邢新, 金华, 董燕 . 玉米ZmSCL7的克隆及功能研究. 中国农业科学, 2013,46:2584-2591.
Guo P, Xing X, Jin H, Dong Y . Cloning and functional study of ZmSCL7 in Zea mays. Sci Agric Sin, 2013,46:2584-2591 (in Chinese with English abstract).
[35] 李慧聪, 李国良, 郭秀林 . 玉米热激转录因子基因ZmHsf-like对逆境胁迫响应的信号途径. 作物学报, 2014,40:622-628.
Li H C, Li G L, Guo X L . Signal transduction pathway of ZmHsf-like gene responding to different abiotic stresses. Acta Agron Sin, 2014,40:622-628 (in Chinese with English abstract).
[36] Klingler J P, Batelli G, Zhu J K . ABA receptors: the START of a new paradigm in phytohormone signaling. J Exp Bot, 2010,61:3199-3210.
doi: 10.1093/jxb/erq151
[37] Fujii H, Verslues P E, Zhu J K . Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo. Proc Natl Acad Sci USA, 2011,108:1717-1722.
[38] Wang R, Jing W, Xiao L Y, Jin Y K, Shen L K, Zhang W H . The rice high-affinity potassium transporter1;1 is involved in salt tolerance and regulated by an MYB-type transcription factor 1. Plant Physiol, 2015,168:1076-1090.
doi: 10.1104/pp.15.00298
[39] Tuberosa R, Sanguineti M C, Landi P, Giuliani M M, Salvi S, Conti S . Identification of QTLs for root characteristics in maize grown in hydroponics and analysis of their overlap with QTLs for grain yield in the field at two water regimes. Plant Mol Biol, 2002,48:697-712.
doi: 10.1023/A:1014897607670
[40] 潘清春 . 两种氮水平下玉米苗期根系与田间产量相关性状QTL定位. 内蒙古农业大学硕士学位论文, 内蒙古呼和浩特, 2010.
Pan Q C . QTL Mapping for Root and Grain Yield Traits in Maize under Two Nitrogen Conditions. MS Thesis of Inner Mongolia Agricultural University, Huhhot, Inner Mongolia, China, 2010.
[1] 肖颖妮, 于永涛, 谢利华, 祁喜涛, 李春艳, 文天祥, 李高科, 胡建广. 基于SNP标记揭示中国鲜食玉米品种的遗传多样性[J]. 作物学报, 2022, 48(6): 1301-1311.
[2] 崔连花, 詹为民, 杨陆浩, 王少瓷, 马文奇, 姜良良, 张艳培, 杨建平, 杨青华. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
[3] 王丹, 周宝元, 马玮, 葛均筑, 丁在松, 李从锋, 赵明. 长江中游双季玉米种植模式周年气候资源分配与利用特征[J]. 作物学报, 2022, 48(6): 1437-1450.
[4] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[5] 陈静, 任佰朝, 赵斌, 刘鹏, 张吉旺. 叶面喷施甜菜碱对不同播期夏玉米产量形成及抗氧化能力的调控[J]. 作物学报, 2022, 48(6): 1502-1515.
[6] 徐田军, 张勇, 赵久然, 王荣焕, 吕天放, 刘月娥, 蔡万涛, 刘宏伟, 陈传永, 王元东. 宜机收籽粒玉米品种冠层结构、光合及灌浆脱水特性[J]. 作物学报, 2022, 48(6): 1526-1536.
[7] 单露英, 李俊, 李亮, 张丽, 王颢潜, 高佳琪, 吴刚, 武玉花, 张秀杰. 转基因玉米NK603基体标准物质研制[J]. 作物学报, 2022, 48(5): 1059-1070.
[8] 朱峥, 王田幸子, 陈悦, 刘玉晴, 燕高伟, 徐珊, 马金姣, 窦世娟, 李莉云, 刘国振. 水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用[J]. 作物学报, 2022, 48(5): 1129-1140.
[9] 陈悦, 孙明哲, 贾博为, 冷月, 孙晓丽. 水稻AP2/ERF转录因子参与逆境胁迫应答的分子机制研究进展[J]. 作物学报, 2022, 48(4): 781-790.
[10] 许静, 高景阳, 李程成, 宋云霞, 董朝沛, 王昭, 李云梦, 栾一凡, 陈甲法, 周子键, 吴建宇. 过表达ZmCIPKHT基因增强植物耐热性[J]. 作物学报, 2022, 48(4): 851-859.
[11] 刘磊, 詹为民, 丁武思, 刘通, 崔连花, 姜良良, 张艳培, 杨建平. 玉米矮化突变体gad39的遗传分析与分子鉴定[J]. 作物学报, 2022, 48(4): 886-895.
[12] 闫宇婷, 宋秋来, 闫超, 刘爽, 张宇辉, 田静芬, 邓钰璇, 马春梅. 连作秸秆还田下玉米氮素积累与氮肥替代效应研究[J]. 作物学报, 2022, 48(4): 962-974.
[13] 徐宁坤, 李冰, 陈晓艳, 魏亚康, 刘子龙, 薛永康, 陈洪宇, 王桂凤. 一个新的玉米Bt2基因突变体的遗传分析和分子鉴定[J]. 作物学报, 2022, 48(3): 572-579.
[14] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[15] 宋仕勤, 杨清龙, 王丹, 吕艳杰, 徐文华, 魏雯雯, 刘小丹, 姚凡云, 曹玉军, 王永军, 王立春. 东北主推玉米品种种子形态及贮藏物质与萌发期耐冷性的关系[J]. 作物学报, 2022, 48(3): 726-738.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2