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作物学报 ›› 2021, Vol. 47 ›› Issue (3): 405-415.doi: 10.3724/SP.J.1006.2021.01049

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

杂交小麦抗坏血酸过氧化物酶基因克隆及其在种子老化中的潜在功能分析

岳洁茹1,2,3(), 白建芳2,3(), 张风廷2,3, 郭丽萍2,3, 苑少华2,3, 李艳梅2,3, 张胜全2,3, 赵昌平2,3,*(), 张立平1,2,3,*()   

  1. 1北京农学院植物科学技术学院, 北京 102206
    2北京市农林科学院北京杂交小麦工程技术研究中心, 北京 100097
    3杂交小麦分子遗传北京市重点实验室, 北京 100097
  • 收稿日期:2020-06-12 接受日期:2020-10-14 出版日期:2021-03-12 网络出版日期:2020-10-28
  • 通讯作者: 赵昌平,张立平
  • 作者简介:岳洁茹, E-mail: 1041705041@qq.com;|白建芳, E-mail: baijianfang1313@yahoo.com
  • 基金资助:
    国家重点研发计划项目(2018YFD0100904);北京市优秀人才项目;北京市农林科学院杰出科学家项目资助(JKZX201907)

Cloning and potential function analysis of ascorbic peroxidase gene of hybrid wheat in seed aging

YUE Jie-Ru1,2,3(), BAI Jian-Fang2,3(), ZHANG Feng-Ting2,3, GUO Li-Ping2,3, YUAN Shao-Hua2,3, LI Yan-Mei2,3, ZHANG Sheng-Quan2,3, ZHAO Chang-Ping2,3,*(), ZHANG Li-Ping1,2,3,*()   

  1. 1College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
    3Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097, China
  • Received:2020-06-12 Accepted:2020-10-14 Published:2021-03-12 Published online:2020-10-28
  • Contact: ZHAO Chang-Ping,ZHANG Li-Ping
  • Supported by:
    National Key Research and Development Program of China(2018YFD0100904);Beijing Excellent Talents Project;Outstanding Scientist Program of Beijing Academy of Agricultural and Forestry Sciences(JKZX201907)

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摘要：

以BS型二系杂交小麦BS1453/11GF5135及其BS1453 (母本)、11GF5135 (父本)的种子为材料, 在BS1453/11GF5135中通过同源克隆获得抗坏血酸过氧化物酶基因TaAPX。该基因包含一个832 bp的ORF, 共编码277个氨基酸。通过进一步生信分析预测miRNA与TaAPX基因的互作关系, 发现TaAPX基因可能受miR396等抗逆及种子活力相关miRNAs的调控。另外通过蛋白互作预测分析, 发现APX蛋白主要与氧化还原相关的酶互作反应。通过对不同老化时间的父母本及杂交种的胚进行qPCR及酶活性分析, 发现TaAPX基因在杂交种及亲本中的表达趋势是先被诱导上调表达, 后下调表达, 但杂交种子内TaAPX基因的表达量在第7天才开始下降, 而亲本种子内TaAPX基因的表达量在第5天开始下降, 且miR396与TaAPX互为拮抗作用。随着老化时间的延长, 亲本种子内APX酶活性表现为下降趋势, 杂交种子内APX酶活性在第3天呈短暂下降趋势, 随后上升, 第9天开始下降, 表明在老化条件下杂交种子内APX酶清除体内过氧化物的能力高于亲本, 即杂交种抗老化能力高于其父母本, 且TaAPX基因对种子活力的调控是一个复杂的多因素参与的调控过程。本研究为进一步研究BS型杂交小麦种子活力的分子调控机理奠定了一定的基础。

关键词: BS型杂交小麦, 人工老化, 抗坏血酸过氧化物酶, 基因克隆, 表达分析

Abstract:

In this study, the seeds of two-line hybrid wheat of BS-type BS1453/11GF5135 and its parents BS1453 (female parent) and 11GF5135 (male parent) were used as materials, and the ascorbate peroxidase gene (TaAPX) was obtained by homologous cloning in BS1453/11GF5135. The gene contains an open reading frame (ORF) of 832 bp, which encoded a total of 277 amino acids. The interaction between miRNAs and TaAPX was predicted by bioinformatics analysis, and the results showed that TaAPX may be regulated by miR396 and other miRNAs that related to stress resistance and seed viability. In addition, it was found that APX protein mainly interacted with redox-related enzymes via protein interaction prediction analysis. qPCR and enzyme activity analysis were conducted on embryos of parents and hybrid at different aging time, it was found that the expression trend of TaAPX gene in hybrid and parents were first up-regulated and then down-regulated. However, the expression of TaAPX gene in hybrid seeds peaked on the seventh day, and it began to decline in parents seeds on the fifth day. There was an antagonistic effect between miR396 and TaAPX. With the increase of aging time, the activity of APX enzyme in parents seeds showed a downtrends, while it showed a temporary downtrends on the third day in hybrid seeds, then it increased and began to decline on the ninth day. It was suggested that the ability of APX enzyme to remove peroxides in the hybrid seeds was higher than that in the parents seeds under the aging condition, that is, the anti-aging ability of hybrid was higher than that of the parents. Moreover, the regulation of seed activity by TaAPX gene was a complex multi-factor regulation process. This study laid a foundation for the further research on the molecular regulation mechanism of the seed vigor of BS type hybrid wheat.

Key words: BS type hybrid wheat, artificial aging, ascorbate peroxidase, gene cloning, expression analysis

表1

TaAPX基因克隆及qPCR引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 退火温度
Annealing
temperature (℃)		 引物用途
Primer purpose
APX-F TATCTCTAGAGGATCCATGGCGGCTCCGGT 71.37 扩增基因引物序列
Amplified gene primer sequences
APX-R TGCTCACCATGGATCCTTACTTGCTCCTCTTGGAAGCCTCG 72.27
APX-qF CAACCGTTGAGTTCATCCCTG 57.8 荧光定量PCR引物序列
RT-qPCR primer sequences
APX-qR TGGTGCGCCTTTCTTAGCAT 60
18S rRNA-qF CGCGCTACGGCTTTGACCTA 56 内参引物序列
Internal reference primer sequences
18S rRNA-qR CGGCAGATTCCCACGCGTTACG 58
miR396-qF CGGTCTTCCACAGCTTTCTT 75 荧光定量PCR引物序列
RT-qPCR primer sequences
miR396-qR GTGCAGGGTCCGAGGT 76
U6snRNA-qF CAAGGATGACACGCAAATTCG 80 内参引物序列
Internal reference primer sequences
U6snRNA-qR GTGCAGGGTCCGAGGT 82.5

图1

亲本及杂交种籽粒形态"

图2

人工老化前后种子萌发表型和发芽统计 A: 老化处理和未老化处理的杂交种及父母本种子的萌发表型。B: 人工老化处理对杂交种及父母本的发芽率、根长、芽长的影响。发芽率在(P<0.01)时差异极显著, 用**表示; 根长在(P < 0.05)时差异显著, 用*表示; 芽长差异不显著。0~5 d: 老化处理的天数。"

图3

TaAPX基因的扩增产物 M: DL2000; 1: BS1453扩增产物; 2: 11GF5135扩增产物; 3: BS1453/11GF5135扩增产物。"

图4

TaAPX氨基酸多序列比对分析"

图5

父母本和杂交种CDS序列比对分析"

图6

TaAPX蛋白的三级结构及磷酸化位点预测 A: TaAPX蛋白的三级结构; B: TaAPX蛋白磷酸化位点的预测。"

图7

TaAPX蛋白系统进化分析"

图8

TaAPX基因与miRNA和蛋白的互作模型及互作位点 A: TaAPX基因与miRNA的互作图; B: miRNA的发卡结构图; C: 与TaAPX蛋白互作的蛋白的网络模型; D: TaAPX基因与miR396的互作位点。"

图9

不同老化时间下TaAPX基因和miR396在杂交种和亲本种子胚中的表达模式分析 A: 不同老化条件下, TaAPX基因和miR396在父本种子胚中的表达; B: 不同老化条件下, TaAPX基因和miR396在母本种子胚中的表达; C: 不同老化条件下, TaAPX基因和miR396在杂交种种子胚中的表达。0~9 d: 老化处理的天数。"

图10

不同老化时间下杂交种和亲本内APX酶活性分析 A: 不同老化条件下, APX酶活在父本种子胚中的变化; B: 不同老化条件下, APX酶活在母本种子胚中的变化; C: 不同老化条件下, APX酶活在杂交种种子胚中的变化。0~9 d: 老化处理的天数。"

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