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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1625-1634.doi: 10.3724/SP.J.1006.2022.11043

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

长链非编码RNA (LncRNA)在印度梨形孢促进大麦根部生长发育中的调控作用

郭楠楠(), 刘天策, 史硕, 胡心亭, 牛亚丹, 李亮()   

  1. 河北工业大学化工学院, 天津 300130
  • 收稿日期:2021-04-13 接受日期:2021-10-19 出版日期:2022-07-12 网络出版日期:2021-11-02
  • 通讯作者: 李亮
  • 作者简介:E-mail: 15033355861@163.com
  • 基金资助:
    国家自然科学基金项目(31801948);河北省重点研发计划项目(19226505D);河北省自然科学基金项目(C2021202005)

Regulation of long non-coding RNA (LncRNA) in barley roots in response to Piriformospora indica colonization

GUO Nan-Nan(), LIU Tian-Ce, SHI Shuo, HU Xin-Ting, NIU Ya-Dan, LI Liang()   

  1. College of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
  • Received:2021-04-13 Accepted:2021-10-19 Published:2022-07-12 Published online:2021-11-02
  • Contact: LI Liang
  • Supported by:
    National Natural Science Foundation of China(31801948);Key Research & Development Projects in Hebei Province(19226505D);Natural Science Foundation of Hebei Province(C2021202005)

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摘要：

印度梨形孢定殖植物会促进植物生物产量提高, 其分子机制有待深入挖掘。LncRNA是一类长链非编码RNA, 在植物生长发育过程中具有重要调控作用。然而, 目前我们还不清楚大麦中LncRNA是否对印度梨形孢的定殖有响应。本研究发现, 印度梨形孢定殖大麦会诱导大麦根系迅速发育, 促进较多根分枝。采用全基因组高通量测序RNA-seq和生物信息学方法鉴定LncRNA, 发现在P. indica定殖后3 d和7 d分别有752个和932个差异表达的LncRNA, 7 d相对于3 d有70个差异表达的LncRNA。其中在P. indica定殖后3 d有375个LncRNA表达上调, 377个LncRNA表达下调; 在P. indica定殖后7 d有459个LncRNA表达上调, 473个LncRNA表达下调; 7 d相对于3 d组中, 有39个LncRNA表达上调, 31个LncRNA表达下调。qPCR验证LncRNA的表达与RNA-seq结果一致。GO和KEGG分析表明, 在P. indica定殖大麦促生过程中, 部分LncRNA参与了激素信号途径的转录调控。该工作对于进一步理解LncRNA与靶基因的相互作用以及其对靶基因的调控功能提供了新的理论基础和实验依据, 并以LncRNA为靶点, 进行作物性状改良提供新的思路和方向。

关键词: LncRNA, 大麦, 印度梨形孢, RNA-seq, 转录因子, 细胞周期

Abstract:

The molecular mechanism of biomass enhancement by Piriformospora indica in colonization plants needs to be further explored. LncRNA is a kind of long-chain non-coding RNA, which plays an important role in the regulation of plant growth and development. However, it remains unclear whether barley LncRNAs are responsive to Piriformospora indica colonization. It was found that barley roots exhibited fast development and large roots branched after P. indica colonization. Genome-wide high throughput RNA-seq and bioinformatical analysis showed that 752 and 932 differentially expressed LncRNAs were detected in responsive to P. indica at 3-day and 7-day after colonization, respectively. And 70 differentially expressed LncRNAs were found at 7-day compared to 3-day. Among these, 375 were up-regulated and 377 were down-regulated after 3 days’ colonization, and 459 were up-regulated and 473 were down-regulated after 7 days’ colonization, 39 were up-regulated and 31 were down-regulated in 7-day to 3-day comparison group. The qPCR results verified the validity of LncRNAs data in RNA-seq. GO and KEGG analysis indicated that a few LncRNAs might be involved in the molecular functions, cellular components, and biological processes upon P. indica colonization. This study provides a new theoretical basis and experimental basis for further understanding of the interaction between LncRNAs and coding sequences and regulatory functional networks, and provides new ideas and directions for crop shape improvement based on LncRNAs.

Key words: LncRNA, barley, Piriformospora indica, RNA-seq, transcription factor, cell cycle

图1

大麦在P. indica定殖后3 d和7 d形态、根长与根数统计结果图 A: 处理组与对照组在3 d的根长和根数, 标尺为 1.0 cm; B: 处理组与对照组在3 d根的分枝情况, 标尺为1.0 cm; C: 处理组与对照组在3 d和7 d的根长统计; D: 处理组与对照组在3 d和7 d的根数统计。进行3个生物学重复实验和统计分析。"

图2

LncRNA 鉴定的高通量测序流程图"

图3

差异表达的LncRNA的层次聚类热图 横坐标为样本名称, 纵坐标为差异表达的LncRNA, 左侧根据表达相似程度对基因进行聚类, 上方根据表达谱的相似程度对每个样本进行聚类, 由蓝至红表达量逐渐上调, 数字为均一化后的相对表达量。"

图4

差异表达LncRNA火山图 A: 3 d vs Mock比较组中差异表达的LncRNA; B: 7 d vs Mock 比较组中差异表达的LncRNA; C: 7 d vs 3 d比较组中差异表达的LncRNA。横坐标表示基因在不同样本或比较组合间的表达倍数变化(log2 (Fold Change)), 横坐标的绝对值越大表明两个比较组合之间的表达变化倍数越大; 纵坐标表示表达差异的显著性水平。表达上调基因用红色点表示, 下调基因用绿色点表示, 蓝色点为未发生显著变化的基因(Padj < 0.05) 。"

表1

用于LncRNA差异表达鉴定的引物"

基因编号
Gene code		 上游引物序列
Forward sequence (5'-3')		 下游引物序列
Reverse sequence (5'-3')
XLOC-070335 TGGCGTCCATATTACGAGGT TTCGGGGAAATATGCTTTTG
XLOC-318641 CATTAGGGAATCCGACGAGA AGTCACGGTTACGCACACAG
XLOC-067560 CCAGTGCAATGATCGCTAGA GGCCTGCTGTCCTTGTTATC
XLOC-119623 TCCGTTTGGTGATTGTGTGT TGCACACGTACAGGGATGAT
XLOC-322519 AGGTAGGTCTCCGTGGGTCT CGAAACCCTCGTTTTGTGTT
XLOC-206343 TCCCCTCTCGTAGATGGACA GACTAAGGAGGGCAGCAAAA
HvUBIQUITIN ACCCTCGCCGACTACAACAT CAGTAGTGGCGGTCGAAGTG

图5

qRT-PCR鉴定LncRNA的差异表达 A: LncRNA_XLOC_070335、XLOC_318641和XLOC_067560基因表达上调; B: LncRNA_XLOC_119623、XLOC_322519和XLOC_206343基因表达下调。qRT-PCR实验和数据分析进行3个生物学重复。*显著差异(P < 0.05)。"

图6

GO富集柱状图 A: 3 d vs Mock组中GO 的富集; B: 7 d vs Mock组中GO的富集; C: 7 d vs 3 d组中GO的富集; 横坐标表示GO条目名称, 分为3类(BP生物学过程, CC细胞组分, MF分子功能)用不同条框区分, 纵坐标为GO条目富集的基因数。"

图7

3个比较组在KEGG途径中富集的差异表达的LncRNA 图中纵坐标代表不同的通路, 横坐标代表相应通路显著差异表达基因占该通路所有基因的比例。圆圈大小代表富集在相应通路中的基因数目, 圆圈越大, 代表富集在该通路中的基因越多。颜色代表富集显著性, 越接近黑色, 代表越显著。"

图8

植物激素信号转导途径"

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