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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 561-569.doi: 10.3724/SP.J.1006.2023.22008

• 研究简报 • 上一篇    下一篇

GsERF6基因过表达对水稻耐盐碱性的影响

才晓溪(), 胡冰霜(), 沈阳, 王研, 陈悦, 孙明哲, 贾博为, 孙晓丽()   

  1. 黑龙江八一农垦大学农学院 / 作物逆境分子生物学实验室, 黑龙江大庆 163319
  • 收稿日期:2022-01-28 接受日期:2022-06-07 出版日期:2022-06-21 网络出版日期:2022-06-21
  • 通讯作者: 孙晓丽
  • 作者简介:才晓溪, E-mail: 18746616279@163.com
    胡冰霜, E-mail: alisa961102@gmail.com第一联系人：

    **同等贡献

  • 基金资助:
    国家自然科学基金项目(32101672);国家自然科学基金项目(31971826);国家自然科学基金项目(U20A2025);中央支持地方高校改革发展资金人才培养支持计划项目(202201005)

Effects of GsERF6 overexpression on salt-alkaline tolerance in rice

CAI Xiao-Xi(), HU Bing-Shuang(), SHEN Yang, WANG Yan, CHEN Yue, SUN Ming-Zhe, JIA Bo-Wei, SUN Xiao-Li()   

  1. Crop Stress Molecular Biology Laboratory, College of Agriculture, Heilongjiang Bayi Agriculture University, Daqing 163319, Heilongjiang, China
  • Received:2022-01-28 Accepted:2022-06-07 Published:2022-06-21 Published online:2022-06-21
  • Contact: SUN Xiao-Li
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(32101672);National Natural Science Foundation of China(31971826);National Natural Science Foundation of China(U20A2025);Special Funds from the Central Finance to Support the Development of Local Universities(202201005)

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摘要：

乙烯响应因子(ERF)是植物特有的一类转录因子, 在响应非生物胁迫中具有重要作用。本研究通过生物信息学发现, 野生大豆耐盐碱ERF转录因子GsERF6与水稻ERF同源蛋白的氨基酸序列相似性很高, 均包含1个高度保守的AP2结构域。为探究GsERF6基因在水稻耐盐碱应答中的作用, 通过遗传转化、PCR和半定量RT-PCR鉴定, 获得了2个纯合的GsERF6过表达转基因水稻株系。表型鉴定表明, 200 mmol L-1 NaHCO3处理下GsERF6转基因水稻的存活率、相对含水量、超氧化物歧化酶、过氧化物酶、过氧化氢酶活性、可溶性糖和脯氨酸含量均显著高于对照, 活性氧积累则反之。实时荧光定量PCR分析表明, 40 mmol L-1 NaHCO3处理6 h后渗透调节基因OsP5CS2和OsLEA14在GsERF6转基因水稻中的表达量显著高于对照。本研究表明, 水稻中GsERF6的过表达可通过提高ROS清除水平、渗透调节能力及胁迫应答基因的表达来提高其耐盐碱性。

关键词: 水稻, 耐盐碱, 乙烯响应因子, 野生大豆, GsERF6

Abstract:

Ethylene response factors (ERFs) are a family of plant specific transcription factors that play important roles in abiotic stress. Bioinformatic analysis revealed that GsERF6, a Glycine soja ERF transcription factor that positively regulated salt-alkaline tolerance, shared high sequence identity with homologous ERF proteins with one highly conserved AP2 domain in rice. To explore the effect of GsERF6 overexpression on the salt-alkaline tolerance in rice, we transformed GsERF6 into rice and obtained two homozygous transgenic lines via PCR and RT-PCR. Phenotypical and physiological assays indicated that, compared with the wild type rice under 200 mmol L-1 NaHCO3 treatment, the survival rate, relative water content, the activities of SOD, POD, and CAT, the soluble sugar and proline contents, were significantly increased, while ROS accumulation was significantly decreased in GsERF6 overexpression lines. The qRT-PCR showed that transcript levels of OsP5CS2 and OsLEA14 were significantly up-regulated in GsERF6 transgenic line after 40 mmol L-1 NaHCO3 treatment for 6 hours. In summary, GsERF6 overexpression in rice contributed to ROS scavenging, osmotic regulation, and activation of stress responsive genes, thus improving the salt-alkaline tolerance of transgenic rice.

Key words: rice, salt-alkaline tolerance, ethylene response factors, Glycine soja, GsERF6

表1

本研究所用的引物"

引物名称
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
GsERF6-F/R GGCTTAAUATGGCTAACGCTGCTG GGTTTAAUTCACACAGCCACGAGCGGT
GsERF6-PCR-F/R ATAAGGAAGTTCATTTCATTTGGA TCTTCGGAACAGCGATTAGCAG
GsERF6-RT-F/R CTGGCCACTCCCCAAAACAAA GAAGGTTCCGAGCCAAACCC
OsElf1-α-F/R GCACGCTCTTCTTGCTTTCAC TCTTGTCAGGGTTGTAGCCGAC
OsP5CS2-F/R GTGGCTTGTGAAGGAGCTGT TTTGACATGCTTTCGTGCTC
OsLEA14-F/R TCGGGATGTCAGGCGATAA GCTTGTAGGTGCTGGTGTCCTT

图1

GsERF6与水稻中同源ERF蛋白进化关系分析 “?”代表水稻ERF基因; “Δ”代表野生大豆ERF基因。"

图2

GsERF6与水稻中同源ERF蛋白序列比对"

图3

GsERF6植物超量表达载体的构建 A: GsERF6过表达载体示意图; B: GsERF6基因的克隆; M: DNA marker; 1~3: GsERF6 PCR产物; C: pC35SU-GsERF6菌落PCR鉴定; M: DNA marker; ?: 阴性H2O对照; +: 阳性质粒对照; 1: pC35SU-GsERF6。"

图4

GsERF6转基因株系的分子检测 A: 转GsERF6基因抗性植株PCR检测; B: 转GsERF6基因植株RT-PCR检测; M: DNA marker; ?: 阴性H2O对照; +: 阳性质粒对照; WT: 野生型对照; #1~#8: 独立株系。"

图5

GsERF6转基因水稻幼苗期耐盐碱性分析 A: NaHCO3处理前后各株系生长状态; B: 水稻幼苗存活率; C: 水稻幼苗相对含水量。差异显著性分析采用t-test方法。"

图6

GsERF6转基因水稻盐碱胁迫下ROS积累及抗氧化物酶活分析 A: DAB染色; B: NBT染色; C: SOD活性; D: POD活性; E: CAT活性。差异显著性分析采用t-test方法。"

图7

GsERF6转基因水稻盐碱胁迫后渗透调节物质含量分析 A: 可溶性糖含量; B: 脯氨酸含量。差异显著性分析采用t-test方法。"

图8

GsERF6转基因水稻盐碱应答基因表达分析 A: OsLEA14基因表达量变化; B: OsP5CS2基因表达量变化。差异显著性分析采用t-test方法。"

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