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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)

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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。"

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