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作物学报 ›› 2019, Vol. 45 ›› Issue (7): 1017-1028.doi: 10.3724/SP.J.1006.2019.84142

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

异源表达棉花S-腺苷甲硫氨酸脱羧酶(GhSAMDC1)基因提高了拟南芥抗盐能力

田文刚1,朱雪峰1,宋雯1,程文翰2,薛飞1,朱华国1,*()   

  1. 1 石河子大学农学院 / 新疆兵团绿洲生态农业重点实验室, 新疆石河子 832003
    2 荆楚理工学院, 湖北荆门 448000
  • 收稿日期:2018-11-05 接受日期:2019-01-19 出版日期:2019-07-12 网络出版日期:2019-03-15
  • 通讯作者: 朱华国
  • 作者简介:E-mail: 631432853@qq.com
  • 基金资助:
    本研究由国家自然科学基金项目(31301363);本研究由国家自然科学基金项目(31660427);新疆生产建设兵团现代农业科技攻关与成果转化计划项目(2015AC007);湖北省自然科学基金项目(2017CFB162)

Ectopic expression of S-adenosylmethionine decarboxylase (GhSAMDC1) in cotton enhances salt tolerance in Arabidopsis thaliana

TIAN Wen-Gang1,ZHU Xue-Feng1,SONG Wen1,CHENG Wen-Han2,XUE Fei1,ZHU Hua-Guo1,*()   

  1. 1 College of Agronomy, Shihezi University / Key Oasis Eco-Agriculture Laboratory of Production and Group, Shihezi 832003, Xinjiang, China
    2 Jingchu University of Technology, Jingmen 448000, Hubei, China
  • Received:2018-11-05 Accepted:2019-01-19 Published:2019-07-12 Published online:2019-03-15
  • Contact: Hua-Guo ZHU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31301363);This study was supported by the National Natural Science Foundation of China(31660427);Science and Technology Development Program of Xinjiang Production and Construction Groups Project(2015AC007);the Natural Science Foundation of Hubei Province(2017CFB162)

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摘要：

以转GhSAMDC1基因拟南芥研究了过量表达GhSAMDC1基因对拟南芥幼苗抗盐能力的影响, 以及内源多胺、过氧化氢(H2O2)、丙二醛(MDA)、叶绿素含量(Chl)、离子渗透率、抗氧化酶(SOD、CAT、POD)活性和表达量在盐胁迫下的变化。结果表明, 过量表达GhSAMDC1基因能够减少拟南芥内源腐胺(Put)含量, 增加亚精胺(Spd)和精胺(Spm)含量。盐胁迫下, 转基因株系亚精胺合酶(AtSPDS1、AtSPDS2)和精胺合酶(AtSPMS)基因表达量明显高于野生型, Spd和Spm含量进一步增加, H2O2、MDA、Chl以及离子渗透率显著降低; 与野生型相比, 过氧化物酶(POD)活力无明显差异, 但超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力明显增加, 其表达水平与活力变化趋势基本一致。因此, 盐胁迫下, GhSAMDC1基因通过提高Spd和Spm合成相关基因的表达, 增加了转基因株系Spd和Spm含量, Spd和Spm直接或间接提高抗氧化系统相关酶的活力, 通过清除H2O2等活性氧的方式提高拟南芥的抗盐能力。

关键词: 拟南芥, 棉花S-腺苷甲硫氨酸脱羧酶基因, 盐胁迫, 抗氧化酶

Abstract:

Transgenic Arabidopsis thaliana (GhSAMDC1) was used to study the effect of overexpression of GhSAMDC1 on salt tolerance of Arabidopsis thaliana seedlings, Contents of endogenous polyamines, hydrogen peroxide (H2O2), malondialdehyde (MDA), and chlorophyll, ion permeability, antioxidant enzymes (SOD, CAT, POD) activities and expression levels were investigated under salt stress. The overexpression of GhSAMDC1 decreased the content of endogenous putrescine (Put) and increased spermidine (Spd) and spermine (Spm) contents in Arabidopsis thaliana. Under salt stress, the expression levels of spermidine synthase (AtSPDS1, AtSPDS2) and spermine synthase (AtSPMS) in transgenic lines were significantly higher than those in wild type, the contents of Spd and Spm were further increased, and the contents of H2O2, MDA, chlorophyll, and ion permeability were obviously decreased. Compared with the wild type, Transgenic lines had no remarkable difference in peroxidase (POD) activity, but significantly higher superoxide dismutase (SOD) and catalase (CAT) activities, with the same change trend as their expression levels. Therefore, GhSAMDC1 increased the contents of Spd and Spm of transgenic plants by increasing the expression of genes related to Spd and Spm synthesis under salt stress, Spd and Spm directly or indirectly increased the activity of enzymes related to antioxidant system, and enhanced the salt tolerance of Arabidopsis thaliana by scavenging H2O2 and other reactive oxygen species.

Key words: Arabidopsis thaliana, cotton S-adenosylmethionine decarboxylase gene, salt stress, antioxidant enzyme

图1

陆地棉GhSAMDC1和其他物种同源蛋白进化树分析及转基因鉴定 A: 陆地棉GhSAMDC1和其他物种同源蛋白进化树分析; B: GhSDAMC1表达载体; C: 转GhSAMDC1基因拟南芥DNA鉴定及表达分析, M、1、2、3、4、5分别为marker、转基因株系1-4、转基因株系1-12、转基因株系1-14、阳性对照、阴性对照, 目的基因片段大小为1035 bp; D: 转GhSAMDC1基因拟南芥表达分析。"

图2

过量表达GhSAMDC1基因对拟南芥内源多胺含量的影响 A~D: 野生型和转基因株系内源的多胺总量、Put、Spd和Spm含量。选取正常培养30 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量, 数据采用Duncan’s法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

附图1

300 mmol L-1 NaCl处理下棉花GhSAMDC1基因表达分析 0、1、3、6、12、24、48、72分别代表棉花幼苗(YZ-1)用300 mmol L-1 NaCl处理0、1、3、6、12、24、48和72 h。培养30 d的棉花幼苗用300 mmol L-1 NaCl处理, 分别在0、1、3、6、12、24、48和72 h取样, 检测不同处理时间段GhSAMDC1基因相对表达量。以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图3

过量表达GhSAMDC1基因对拟南芥抗盐能力的影响 A: 种子分布示意图; B~C: 正常和盐胁迫下培养15 d后野生型及转基因拟南芥表型; D~E: 正常培养15 d后野生型和转基因拟南芥叶片数及鲜重; F~H: 100 mmol L-1 NaCl处理15 d后野生型和转基因拟南芥成活率、鲜重及叶片数。野生型和转基因拟南芥在正常培养和100 mmol L-1 NaCl处理15 d后统计鲜重、叶片数和成活率。数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准误表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图4

盐胁迫下过量表达GhSAMDC1基因对拟南芥叶绿素含量影响 A~C: 盐胁迫下野生型和转基因株系叶绿素含量。选取100 mmol L-1 NaCl处理15 d的拟南芥叶片, 检测叶绿素a和叶绿素b含量。数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图5

盐胁迫下过量表达GhSAMDC1基因对拟南芥内源多胺含量的影响 A~D: 正常和盐胁迫下野生型及转基因株系内源的多胺总量、Put、Spd和Spm含量对比。选取正常培养和100 mmol L-1 NaCl处理15 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量。数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图6

盐胁迫下过量表达GhSAMDC1基因对拟南芥内源基因表达的影响 A~C: 野生型和转基因拟南芥AtSPDS1、AtSPDS2和AtSPMS相对表达量。选取100 mmol L-1 NaCl处理15 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量, 数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图7

盐胁迫下过量表达GhSAMDC1基因对拟南芥叶片H2O2、MDA及离子渗透率影响 A: 盐胁迫下野生型和转基因株系DAB染色; B~D: 盐胁迫下野生型和转基因株系H2O2、MDA含量及离子渗透率; 选取100 mmol L-1 NaCl处理15 d的拟南芥叶片, 分别进行DAB染色, H2O2、MDA含量以及离子渗透率的检测, 数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

图8

盐胁迫下过量表达GhSAMDC1基因对拟南芥抗氧化酶活力和表达的影响 A~C: 盐胁迫下野生型和转基因株系CAT、SOD、POD活力; D~F: 盐胁迫下野生型和转基因株系CAT、SOD、POD表达分析。选取100 mmol L-1 NaCl处理15 d的拟南芥叶片, 分别检测CAT、SOD和POD的活力和相对表达量。数据采用Duncan法进行差异显著性检验(* P < 0.05, ** P < 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。"

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