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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)

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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。"

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