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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1625-1634.doi: 10.3724/SP.J.1006.2022.11043

• OCROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-07-12 Published:2021-11-02
  • Contact: LI Liang E-mail:15033355861@163.com;liangli@hebut.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31801948);Key Research & Development Projects in Hebei Province(19226505D);Natural Science Foundation of Hebei Province(C2021202005)

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

Fig. 1

Barley root physio morphology, root length, and root number at 3 days and 7 days after P. indica colonization P. indica colonization A: root length and root number were compared between treatment and Mock at 3 days. Bar: 1.0 cm; B: branched roots were compared between treatment and Mock at 3 days. Bar: 1.0 cm; C: chart showing the statistical results of the root length of barley at 3 days and 7 days; D: chart showing the statistical results of the root number of barley at 3 days and 7 days. The experiments and statistical analysis were performed in three biological replicates."

Fig. 2

High-throughput sequencing flowchart of LncRNA identification"

Fig. 3

Hierarchical clustering heat map of differentially expressed LncRNA The abscissa is the sample, the ordinate is the differentially expressed LncRNA. On the left side, the genes are clustered according to the degree of similarity of expression, and the upper part of the sample is clustered according to the degree of similarity of the expression profile. The relative expression level is gradually up-regulated from blue to red, and the number is the relative expression after standardization."

Fig. 4

Differentially expressed LncRNA volcano map A: volcano map of differentially expressed LncRNA in 3 days vs Mock comparison group; B: volcano map of differentially expressed LncRNA in 7 days vs Mock comparison group; C: volcano map of differentially expressed LncRNA in 7 days vs 3 days comparison group. The abscissa indicates the multiple change of gene expression between different samples or comparison combinations (log2 (Fold Change)). The larger the absolute value of abscissa indicates the greater the multiple of expression change between the two comparison combinations; the ordinate indicates the significant level of expression difference. The up-regulated genes are represented by red dots, the down-regulated genes are represented by green dots, and the blue dots are genes that have not changed significantly (Padj < 0.05)."

Table 1

Primers for differential expression identification of LncRNA"

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

Fig. 5

Identification of differential expression of LncRNA by qRT-PCR A: LncRNA_XLOC_070335, XLOC_318641, and XLOC_067560 were up-regulated; B: LncRNA_XLOC_119623, XLOC_322519, and XLOC_206343 were down-regulated. The experiments of qRT-PCR and the data analysis were performed in three biological replicates. *: P < 0.05."

Fig. 6

GO enrichment histogram A: GO enrichment in 3 days vs Mock group; B: GO enrichment in 7 days vs Mock group; C: GO enrichment in 7 days vs 3 days group. The abscissa denotes the name of GO entry, which is divided into three categories by box (BP biological process, CC cell component, MF molecular function), distinguished by different frames, and the ordinate is the number of genes enriched by GO entry."

Fig. 7

Differentially expressed LncRNA enriched in KEEG pathway in three comparison groups The ordinate represents different pathways, and the abscissa represents the proportion of differentially expressed genes in the corresponding pathway to all genes in the pathway. The circle size represents the number of genes enriched in the corresponding pathway, and the larger the circle, the more genes are enriched in the pathway. Color represents enrichment significance, and the closer it is to be black, the more significant it is."

Fig. 8

Plant hormone signal transduction"

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