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作物学报 ›› 2021, Vol. 47 ›› Issue (8): 1417-1426.doi: 10.3724/SP.J.1006.2021.01071

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

小麦lncRNA27195及其靶基因TaRTS克隆及表达分析

王娜1,2(), 白建芳2, 马有志1, 郭昊宇2, 王永波2, 陈兆波2, 赵昌平2,*(), 张立平2,*()   

  1. 1中国农业科学院作物科学研究所, 北京 100087
    2北京市农林科学院北京杂交小麦工程技术研究中心/杂交小麦分子遗传北京市重点实验室, 北京 100097
  • 收稿日期:2020-09-02 接受日期:2021-01-13 出版日期:2021-08-12 网络出版日期:2021-02-22
  • 通讯作者: 赵昌平,张立平
  • 作者简介:王娜, E-mail: wangna_00@sina.com
  • 基金资助:
    北京市自然科学基金项目(6182014);北京市农林科学院杰出科学家项目(JKZX201907);北京市农林科学院青年基金(QNJJ201916);国家自然科学基金项目(31872881);北京市农林科学院科技创新能力建设专项(KJCX20180403)

Cloning and expression analysis of lncRNA27195 and its target gene TaRTS in wheat (Triticum aestivum L.)

WANG Na1,2(), BAI Jian-Fang2, MA You-Zhi1, GUO Hao-Yu2, WANG Yong-Bo2, CHEN Zhao-Bo2, ZHAO Chang-Ping2,*(), ZHANG Ling-Ping2,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100087, China
    2Beijing Engineering and Technique Research Center for Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences/Municipal Key Laboratory of the Molecular Genetics of Hybrid Wheat, Beijing 100097, China
  • Received:2020-09-02 Accepted:2021-01-13 Published:2021-08-12 Published online:2021-02-22
  • Contact: ZHAO Chang-Ping,ZHANG Ling-Ping
  • Supported by:
    Natural Science Foundation of Beijing(6182014);Outstanding Scientist Cultivation Program of BAAFS(JKZX201907);Foundation for Youths of BAAFS(QNJJ201916);National Natural Science Foundation of China(31872881);Special Project of Science and Technology Innovation Ability Construction of BAAFS(KJCX20180403)

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摘要：

长链非编码RNA (long non-coding RNA, lncRNA)是一种大于200 bp的非编码RNA, 大量存在于植物体中, 其可通过调节基因表达或蛋白功能, 在植物生长发育与胁迫应答中发挥重要作用。前期在研究光照和温度对小麦光温敏核雄性不育系BS366育性诱导中, 利用转录组测序筛选获得与育性相关的lncRNA (lncRNA2719)。为研究小麦lncRNA27195的功能, 本研究在BS366中克隆lncRNA27195及其靶基因TaRTS, 并对TaRTS进行生物信息学分析, 结果显示, TaRTS基因全长315 bp, 编码104个氨基酸, 且发现该RTS蛋白仅存在于禾本科植物中。通过实时荧光定量PCR, 对lncRNA27195及TaRTS基因在不同组织不同光温处理及茉莉酸甲酯处理下进行表达模式分析, 发现lncRNA27195和TaRTS均在雄蕊中高表达, 呈显著的正相关, 且两者在不同光温条件下呈现出不同的表达模式。光照和温度均对lncRNA27195和TaRTS有调控作用, 适当浓度的MeJA促进两者的表达, SA抑制两者表达。以上结果表明, 在光周期、温度和植物激素的诱导下, lncRNA27195正向调节TaRTS基因表达, 进而影响花粉育性, 本研究结果有助于促进对小麦光温敏核型雄性不育系的机理研究和生产应用。

关键词: lncRNA, RTS蛋白, 花药, 雄性不育

Abstract:

Long non-coding RNA (lncRNA) is a non-coding RNA length over 200 bp, which is abundant in plants. It plays important roles in plant growth, development, and stress response by regulating gene expression or protein function. In the previous study, a fertility-related lncRNA named lncRNA27195 was screened and obtained by transcriptome sequencing from the anther of wheat Photoperiod-thermo Sensitive Genic Male Sterility (PTGMS) line BS366. To investigate the function of lncRNA27195 in wheat, the lncRNA27195 gene and its target gene TaRTS were cloned from BS366. Bioinformatics analysis were performed on TaRTS. The expressions of lncRNA27195 and TaRTS in different tissues and their expression correlation between them were analyzed by qRT-PCR. Meanwhile, the expression patterns of lncRNA27195 and TaRTS under different light and temperature treatments, and methyl jasmonate (MeJA) treatments were investigated. The results showed that the TaRTS gene with 315 bp length, encoded 104 amino acids. Additionally, RTS proteins were only found as anther-specific proteins in gramineae plants. Both lncRNA27195 and TaRTS with a significantly positive correlation were highly expressed in stamens, and revealed different expression patterns in different fertility environments. The results demonstrated that the expression of lncRNA27195 and TaRTS were also regulated by light and temperature. In addition, we found that the appropriate concentration of MeJA could promote the expression of lncRNA27195 and TaRTS while SA could inhibit the expression. The results indicated that under the induction of photoperiod, temperature, and plant hormones, IncRNA27195 positively regulated TaRTS gene expression, resulting in affecting pollen development and male fertility. This study contributed to the mechanism research and production application of PTGMS wheat.

Key words: lncRNA, RTS protein, anther, male sterility

表1

引物设计"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 产物大小
Product length (bp)
lncRNA27195-F TTTAATCTTCCGAGCTCACCCA 396
lncRNA27195-R TTTAAATGTTAAAACATGTTATATTTTTGA
TaRTS-F TTTAAATGGCACGCACCGGC 315
TaRTS-R TTTAATCAGAGCCCCTGGGTGGT
lncRNA27195-qRT-F GCATACGCACCGCATGTATAAT 109
lncRNA27195-qRT-R CTAGTCGCACAGGTCGACTGAA
TaRTS-qRT-F AGGTCGCATCGTGCCTCCT 114
TaRTS-qRT-R CCCCTGGGTGGTGCAGT
Actin1-F TTGGCATCTCTCAGCACCTTC 138
Actin1-R TCAGCCTTCGCAATCCACA

图1

lncRNA27195基因和TaRTS基因扩增片段(A)和序列(B, C)"

图2

lncRNA27195基因(A)和TaRTS基因(B)序列比对"

图3

TaRTS蛋白的亲水性(A)、二级结构(B)和三级结构(C) 蓝色为α-螺旋, 绿色为β-转角, 红色为延伸链, 紫色为无规则卷曲。"

图4

TaRTS蛋白的小麦原生质体亚细胞定位"

图5

TaRTS的多序列比对(A)和进化树分析(B)"

表2

启动子元件分析"

上游启动子元件 Upstream promoter elements 功能 Function
CAAT-box, TATA-box 核心启动子元件 Core promoter element
ACE, G-box, GT1-motif, T-box, Sp1 光响应元件 Light responsiveness element
ABRE 脱落酸响应元件 Abscisic acid responsiveness element
CAT-box 分生组织表达相关元件 Related to meristem expression element
CCAAT-box MYBHv1结合位点 MYBHv1 binding site
CGTCA-motif, TGACG-motif 茉莉酸甲酯响应元件 MeJA-responsiveness responsiveness element
LTR 低温响应元件 Low-temperature responsiveness element
MSA-like 细胞周期调控元件 Cell cycle regulation element

图6

lncRNA27195基因(A)及TaRTS基因(B)组织特异性表达分析"

图7

TaRTS(A)和lncRNA27195(B)表达模式分析及两者表达量相关性分析(C) *代表显著差异(P<0.05), **代表极显著差异(P<0.01)。"

图8

TaRTS及lncRNA27195在不同光照温度条件下表达模式分析 T1: 温长日环境(20℃, 14 h L/10 h D); T2: 高温短日环境(20℃, 12 h L/12 h D); T3: 低温长日环境(12℃, 14 h L/10 h D); T4为低温短日(12℃, 12 h L/12 h D)环境。*代表各组间存在显著差异(P < 0.05)。"

图9

TaRTS及lncRNA27195在MeJA和SA处理下的表达模式分析"

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