欢迎访问作物学报,今天是

作物学报 ›› 2019, Vol. 45 ›› Issue (9): 1311-1318.doi: 10.3724/SP.J.1006.2019.81053

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦抗逆相关基因TaSAP1的5′非翻译区内含子功能分析

常建忠1,董春林1,张正1,乔麟轶2,杨睿1,蒋丹1,张彦琴1,杨丽莉1,吴佳洁3,景蕊莲4,*()   

  1. 1 山西省农业科学院旱地农业研究中心, 山西太原 030031
    2 山西省农业科学院作物科学研究所, 山西太原 030031
    3 山东农业大学/作物生物学国家重点实验室, 山东泰安 271018
    4 农作物基因资源与基因改良国家重大科学工程/中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2018-12-12 接受日期:2019-05-12 出版日期:2019-09-12 网络出版日期:2019-05-17
  • 通讯作者: 景蕊莲
  • 作者简介:E-mail: cjzyfx@163.com
  • 基金资助:
    本研究由国家自然科学基金项目(31401385);山西省自然科学基金重点基金项目(201601D011001);山西省农业科学院优势课题组自选项目资助(YYS1705)

Function analysis of 5′ untranslated region introns in drought-resistance gene TaSAP1

CHANG Jian-Zhong1,DONG Chun-Lin1,ZHANG Zheng1,QIAO Lin-Yi2,YANG Rui1,JIANG Dan1,ZHANG Yan-Qin1,YANG Li-Li1,WU Jia-Jie3,JING Rui-Lian4,*()   

  1. 1 Dryland Agriculture Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    2 Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    3 Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong, China;
    4 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-12-12 Accepted:2019-05-12 Published:2019-09-12 Published online:2019-05-17
  • Contact: Rui-Lian JING
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31401385);the Key Project of Natural Science Foundation of Shanxi Province(201601D011001);the Optional Project of Advantage Research Group of Shanxi Academy of Agricultural Sciences(YYS1705)

摘要:

逆境相关蛋白(stress associated protein, SAP)是植物中一类具有A20/AN1锌指结构域的蛋白, 而A20/AN1锌指蛋白主要参与植物逆境响应。小麦中TaSAP1基因参与植株对各种逆境的响应, 其5′非翻译区含有2个内含子(5' untranslated region intron, 5UI)。本研究利用重叠PCR, 构建了TaSAP1的2个5UI缺失突变的表达载体, 并转化二穗短柄草(Brachypodium distachyon), 通过分析GUS活性, 研究TaSAP1启动子及其5UI的生物学功能。结果表明TaSAP1启动子P1可受干旱、低温和外源ABA胁迫上调表达, 表达活性分别是对照的10、6和4倍。TaSAP1基因2个5UI对启动子活性至关重要, 2个5UI同时突变可致P1失活; Intron-1缺失突变导致P1活性降低约2.7倍(P<0.05), 但不影响P1对逆境胁迫的响应; Intron-2缺失突变导致P1失去对干旱、低温和外源ABA的响应能力。本研究结果为深入研究TaSAP1 5UI的生物学功能奠定了基础, 也为改善作物抗逆性提供了重要元件。

关键词: 小麦, 5′非翻译区内含子, 启动子, GUS定量分析

Abstract:

Stress associated proteins (SAPs), a group of A20/AN1 zinc-finger domain-containing proteins, are mainly involved in responding to abiotic stresses in plant. TaSAP1 involved in the responses of wheat to several abiotic stresses has two introns in the 5' untranslated region intron (5UIs). In this study, the two 5UIs were removed respectively by using overlapping PCR, and the expression vectors were constructed and transformed into Brachypodium distachyon via Agrobacterium-mediated transformation. The functions of TaSAP1 promoter and 5UIs were analyzed according to GUS activity. The activity of TaSAP1 promoter (P1) was up-regulated under the stresses of drought, cold and exogenous abscisic acid (ABA) by 10, 6, and 4 folds, respectively. The 5UIs were absolutely necessary for the promoter activity that lost in the double mutants of 5UIs. Intron-1 deletion led to 2.7-fold decrease (P<0.05) in GUS activity, whereas Intron-2 deletion resulted in that P1 was not able to respond to drought, low temperature and exogenous abscisic acid. These results provide basic information for further study in biological function of TaSAP1 5UIs.

Key words: wheat, 5′ untranslated region introns, promoter, quantitative GUS assay

图1

TaSAP1 5′UTR内含子缺失突变示意图"

图2

过表达载体构建示意图"

图3

TaSAP1启动子序列及其顺式作用元件分布 顺式元件用红色背景显示。"

表1

TaSAP1启动子主要顺式作用元件及预测的功能"

元件名称
Name
序列
Sequence
功能
Function
位置
Location (bp)
ABRE GACACGTGGC/TACGTG ABA响应
cis-acting element involved in abscisic acid responsivenes
-937 to -926,
-510 to -504
ARE AAACCA 低氧响应
cis-acting regulatory element essential for anaerobic induction
-1256 to -1249
Box-W1 GGTCAA 真菌诱导响应
Fungal elicitor responsive element
-2062 to -2055
CGTCA-motif CGTCA 茉莉酸甲酯响应
cis-acting regulatory element involved in MeJA-responsiveness
-2107 to -2101
CCAAT-box CCGTTG MYB转录因子结合元件
MYB binding site
-1176 to -1169,
-658 to -651
GC-motif CGGGGGC 低氧特异增强元件
Enhancer-like element involved in anoxic specific inducibility
-1534 to -1526
LTR CCGAAA 低温响应
cis-acting element involved in low-temperature responsiveness
-1233 to -1226,
-1207 to -2199
MBS TGACCGA MYB转录因子结合元件
MYB binding site
-1448 to -1440,
-39 to -32
Skn-1 motif GTCAT 胚乳特异表达元件
cis-acting regulatory element required for endosperm expression
-1323 to -1317,
-1108 to -1102
TGACG-motif TGACG 茉莉酸甲酯响应
cis-acting regulatory element involved in MeJA-responsiveness
-1914 to -1908
WUN-motif TCATTGCGAA 伤害响应
Wound-responsive element
-2103 to -2092

图4

TaSAP1 5UI缺失突变及载体构建 M: marker III (天根); A: 分别用TaspF1/R1 (泳道1)和TaspF2/R2 (泳道2)进行第1轮PCR扩增; B: 用TaspF1/R2进行第2轮PCR扩增, 缺失Intron-1; C: 用TspF1/R3进行PCR扩增获得-2I片段(泳道1); 用TspF1/R3扩增获得Δ2I片段(泳道2); D: 1 ~ 4泳道依次为载体P91z-P1、P91z-(-1I)、P91z-(-2I)和P91z-Δ2I的酶切验证。"

图5

二穗短柄草的遗传转化 A: 短柄草幼胚分化的愈伤组织; B: 筛选中褐化的胚型愈伤组织; C和D: 再生的转基因植株; E和F: 移栽后的转基因植株。"

图6

转基因阳性二穗短柄草植株GUS染色及定量分析 A: 转基因阳性(上排)和非转基因(下排)二穗短柄草GUS染色分析。从左至右依次为叶片、幼穗、颖壳、种子、幼茎和幼根。B: 转基因阳性二穗短柄草GUS定量分析。*表示两者之间差异在0.05水平上显著(t检验)。"

图7

TaSAP1启动子及其内含子缺失突变体对不同逆境的响应 A: 胁迫条件下转基因二穗短柄草叶片的GUS组织化学染色; B: 胁迫条件下转基因二穗短柄草的GUS定量分析。NaCl、PEG、4°C和ABA表示生长2周后的二穗短柄草分别在NaCl (250 mmol L-1)、PEG-6000 (-0.5 MPa)、4°C和ABA (50 μmol L-1)条件下处理8 h, CK为无处理对照。*和**分别表示胁迫处理与对照(CK)的酶活差异在0.05和0.01水平上显著。"

[1] Opipari A W Jr, Boguski M S, Dixit V M . The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J Biol Chem, 1990,265:14705-14708.
[2] Linnen J M, Bailey C P, Weeks D L . Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins. Gene, 1993,128:181-188.
[3] Beyaert R, Heyninck K, Van Huffel S . A20 and A20-binding proteins as cellular inhibitors of nuclear factor-kB-dependent gene expression and apoptosis. Biochem Pharmacol, 2000,60:1143-1151.
[4] Kanneganti V, Gupta A K . Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice. Plant Mol Biol, 2008,66:445-462.
[5] Gimeno-Gilles C, Gervais M L, Planchet E, Satour P, Limami A M, Lelievre E . A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds. Plant Physiol Biochem, 2011,49:303-310.
[6] Kang M, Lee S, Abdelmageed H, Reichert A, Lee H K, Fokar M, Mysore K S, Allen R D . Arabidopsis stress associated protein 9 mediates biotic and abiotic stress responsive ABA signaling via the proteasome pathway. Plant Cell Environ, 2017,40:702-716.
[7] Dixit A, Tomar P, Vaine E, Abdullah H, Hazen S, Dhankher O P . A stress-associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses. Plant Cell Environ, 2018,41:1171-1185.
[8] Ghneim-Herrera T, Selvaraj M G, Meynard D, Fabre D, Pena A, Ben Romdhane W, Ben Saad R, Ogawa S, Rebolledo M C, Ishitani M, Tohme J, Al-Doss A, Guiderdoni E, Hassairi A . Expression of the Aeluropus littoralis AlSAP gene enhances rice yield under field drought at the reproductive stage. Front Plant Sci, 2017,8:994.
[9] Gruss P, Lai C J, Dhar R, Khoury G . Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci USA, 1979,76:4317-4321
[10] Le Hir H, Nott A, Moore M J . How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci 2003, 28:215-220.
[11] Callis J, Fromm M, Walbot V . Introns increase gene expression in cultured maize cells. Genes Dev, 1987,1:1183-1200.
[12] Clancy M, Hannah L C . Splicing of the maize Sh1 first intron is essential for enhancement of gene expression, and a T-rich motif increases expression without affecting splicing. Plant Physiol, 2002,130:918-929.
[13] Donath M, Mendel R, Cerff R, Martin W . Intron-dependent transient expression of the maize GapA1 gene. Plant Mol Biol, 1995,28:667-676
[14] Sinibaldi R M, Mettler I J . Intron splicing and intron-mediated enhanced expression in monocots. Prog Nucl Acid Res Mol Biol, 1992,42:229-257.
[15] Wang Y, Lang Z, Zhang J, He K, Song F, Huang D . Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize(Zea mays L.). Chin Sci Bull, 2008,53:3185-3190.
[16] 陈俊, 王宗阳 . 水稻OsBP-73基因表达需要其内含子参与. 植物生理和分子生物学学报, 2004,30:81-86.
Chen J, Wang Z Y . Expression of OsBP-73 gene requires involvement of its intron in rice. J Plant Physiol Mol Biol, 2004,30:81-86 (in Chinese with English abstract).
[17] Giani S, Altana A, Campanoni P, Morello L, Breviario D . In trangenic rice, alpha- and beta-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression. Transgenic Res, 2009,18:151-162.
[18] Samadder P, Sivamani E, Lu J, Li X, Qu R . Transcriptional and post-transcriptional enhancement of gene expression by the 5° UTR intron of rice rubi3 gene in transgenic rice cells. Mol Genet Genomics, 2008,279:429-439.
[19] Akua T, Shaul O . The Arabidopsis thaliana MHX gene includes an intronic element that boosts translation when localized in a 5° UTR intron. J Exp Bot, 2013,64:4255-4270.
[20] Gallois J L, Drouaud J, Lecureuil A, Guyon-Debast A, Bonhomme S, Guerche P . Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana. Gene, 2013,526:299-308.
[21] Mufarrege E F, Gonzalez D H, Curi G C . Functional interconnections of Arabidopsis exon junction complex proteins and genes at multiple steps of gene expression. J Exp Bot, 2011,62:5025-5036.
[22] Bartlett Joanne G, Snape J W, Harwood W A . Intron-mediated enhancement as a method for increasing transgene expression levels in barley. Plant Biotechnol J, 2009,7:856-866.
[23] Jin Y, Wang M, Fu J, Xuan N, Zhu Y, Lian Y, Jia Z, Zheng J, Wang G . Phylogenetic and expression analysis of ZnF-AN1 genes in plants. Genomics, 2007,90:265-275.
[24] Ben Saad R, Zouari N, Ben Ramdhan W, Azaza J, Meynard D, Guiderdoni E, Hassairi A . Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “ AlSAP ” gene isolated from the halophyte grass Aeluropus littoralis. Plant Mol Biol, 2010,72:171-190.
[25] Sreedharan S, Shekhawat U K, Ganapathi T R . MusaSAP1, a A20/AN1 zinc finger gene from banana functions as a positive regulator in different stress responses. Plant Mol Biol, 2012,80:503-517.
[26] 王彩香 . 小麦抗逆相关基因 TaABC1TaSAP1/2 的分离及功能分析. 中国农业科学院博士学位论文, 北京, 2011.
Wang C X . Isolation and Functional Analysis of Stress-response Genes TaABC1 and TaSAP1/2 from Wheat (Triticum aestivum L.). PhD Dissertation of Chinese Academy of Argicultural Sciences, Beijing, China, 2011 (in Chinese with English abstract).
[27] Chang J, Zhang J, Mao X, Li A, Jia J, Jing R . Polymorphism of TaSAP1-A1 and its association with agronomic traits in wheat. Planta, 2013,237:1495-1508.
[28] Bragg J N, Wu J, Gordon S P, Guttman M E, Thilmony R, Lazo G R, Gu Y Q, Vogel J P . Generation and characterization of the western regional research center Brachypodium T-DNA insertional mutant collection. PLoS One, 2012,7:e41916.
[29] Jefferson R A . Assaying chimeric genes in plants: the gus gene fusion system. Plant Mol Biol Rep, 1987,1987:387-405.
[30] 雷建峰, 伍娟, 陈晓俊, 於添平, 倪志勇, 李月, 张巨松, 刘晓东 . 棉花花粉中高效转录U6启动子的克隆及功能分析. 中国农业科学, 2015,48:3794-3802.
Lei J F, Wu J, Chen X J, Yu T P, Ni Z Y, Li Y, Zhang J S, Liu X D . Cloning and functional analysis of cotton U6 promoter with high transcription activity in cotton pollen. Sci Agric Sin, 2015,48:3794-3802 (in Chinese with English abstract).
[31] 扆珩, 李昂, 刘惠民, 景蕊莲 . 小麦蛋白磷酸酶2A基因TaPP2AbB”-α启动子的克隆及表达分析. 作物学报, 2016,42:1282-1290.
Yi H, Li A, Liu H M, Jing R L . Cloning and expression analysis of protein phosphatase 2A gene TaPP2AbB”-α promoter in wheat. Acta Agron Sin, 2016,42:1282-1290 (in Chinese with English abstract).
[32] Cenik C, Derti A, Mellor J C, Berriz G F, Roth F P . Genome-wide functional analysis of human 5° untranslated region introns. Genome Biol, 2010,11:R29.
[33] 谢先芝, 吴乃虎 . 番茄蛋白酶抑制剂Ⅱ基因的分离及其内含子功能. 科学通报, 2001,46:934-938.
Xie X Z, Wu N H . Isolation and functional analysis of proteinase inhibitor gene in tomato. Chin Sci Bull, 2001,46:934-938 (in Chinese).
[34] 焦博, 柏峰, 李艳艳, 路佳, 张肖, 曹艺茹, 葛荣朝, 赵宝存 . 耐盐小麦中TaSC基因启动子的克隆及调控功能分析. 作物学报, 2018,44:620-626.
Jiao B, Baifeng, Li Y Y, Lu J, Zhang X, Cao Y R, Ge R C, Zhao B C . Cloning and regulation function analysis of TaSC promoter fromsalt tolerant wheat. Acta Agron Sin, 2018,44:620-626 (in Chinese with English abstract).
[35] Schledzewski K, Mendel R R . Quantitative transient gene expression: comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derived Emu and CaMV35S in cells of barley, maize and tobacco. Transgenic Res, 1994,3:249-255.
[36] 叶兴国 . 新模式植物短柄草模式特性研究进展. 作物学报, 2008: 34:919-925.
Ye X G . Research outline on some related characteristics of Brachypodium distachyon as a new model plant species. Acta Agron Sin, 2008,34:919-925 (in Chinese with English abstract).
[1] 胡文静, 李东升, 裔新, 张春梅, 张勇. 小麦穗部性状和株高的QTL定位及育种标记开发和验证[J]. 作物学报, 2022, 48(6): 1346-1356.
[2] 郭星宇, 刘朋召, 王瑞, 王小利, 李军. 旱地冬小麦产量、氮肥利用率及土壤氮素平衡对降水年型与施氮量的响应[J]. 作物学报, 2022, 48(5): 1262-1272.
[3] 付美玉, 熊宏春, 周春云, 郭会君, 谢永盾, 赵林姝, 古佳玉, 赵世荣, 丁玉萍, 徐延浩, 刘录祥. 小麦矮秆突变体je0098的遗传分析与其矮秆基因定位[J]. 作物学报, 2022, 48(3): 580-589.
[4] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[5] 冯健超, 许倍铭, 江薛丽, 胡海洲, 马英, 王晨阳, 王永华, 马冬云. 小麦籽粒不同层次酚类物质与抗氧化活性差异及氮肥调控效应[J]. 作物学报, 2022, 48(3): 704-715.
[6] 刘运景, 郑飞娜, 张秀, 初金鹏, 于海涛, 代兴龙, 贺明荣. 宽幅播种对强筋小麦籽粒产量、品质和氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 716-725.
[7] 马红勃, 刘东涛, 冯国华, 王静, 朱雪成, 张会云, 刘静, 刘立伟, 易媛. 黄淮麦区Fhb1基因的育种应用[J]. 作物学报, 2022, 48(3): 747-758.
[8] 徐龙龙, 殷文, 胡发龙, 范虹, 樊志龙, 赵财, 于爱忠, 柴强. 水氮减量对地膜玉米免耕轮作小麦主要光合生理参数的影响[J]. 作物学报, 2022, 48(2): 437-447.
[9] 王洋洋, 贺利, 任德超, 段剑钊, 胡新, 刘万代, 郭天财, 王永华, 冯伟. 基于主成分-聚类分析的不同水分冬小麦晚霜冻害评价[J]. 作物学报, 2022, 48(2): 448-462.
[10] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[11] 马博闻, 李庆, 蔡剑, 周琴, 黄梅, 戴廷波, 王笑, 姜东. 花前渍水锻炼调控花后小麦耐渍性的生理机制研究[J]. 作物学报, 2022, 48(1): 151-164.
[12] 孟颖, 邢蕾蕾, 曹晓红, 郭光艳, 柴建芳, 秘彩莉. 小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能[J]. 作物学报, 2022, 48(1): 63-75.
[13] 韦一昊, 于美琴, 张晓娇, 王露露, 张志勇, 马新明, 李会强, 王小纯. 小麦谷氨酰胺合成酶基因可变剪接分析[J]. 作物学报, 2022, 48(1): 40-47.
[14] 李玲红, 张哲, 陈永明, 尤明山, 倪中福, 邢界文. 普通小麦颖壳蜡质缺失突变体glossy1的转录组分析[J]. 作物学报, 2022, 48(1): 48-62.
[15] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!