Identification and functional analysis of lncRNA167 and its cleavage product miR167c in Medicago truncatula

doi:10.11686/cyxb2020501

Abstract

Abstract:

Long non-coding RNA （lncRNA） is a class of functional RNA molecules with a length of more than 200 nucleotides， which plays important roles in various biological regulation processes and response to biological and abiotic stresses. lncRNAs often interacts with microRNAs （miRNAs）. In this study， a salt stress induced lncRNA （Mt-lncRNA167） obtained by high-throughput sequencing was identified to be a precursor of miRNA. The chromosome location of Mt-lncRNA167 coincided with that of miR167c， a member of miR167 family in Medicago truncatula. Mt-lncRNA167 was predicted to play a role as a cleavage precursor of Mt-miR167c. The online software analysis showed that auxin response factor 6/8 （ARF6/8） were two target genes of Mt-miR167c. The expression pattern analysis showed that Mt-lncRNA167 mainly expressed in leaves. Mt-lncRNA167 and Mt-miR167c responded to 100 mmol·L^-1 NaCl， 5% PEG 6000， and 4 ℃ cold stress， which was consistent with the prediction of their promoter cis-acting elements. Under salt， drought and low temperature stresses， the expression patterns of Mt-lncRNA167 and Mt-miR167c were almost the same， which were contrary to the expression patterns of target genes ARF6/8. Mt-lncRNA167 and Mt-miR167c overexpressing Arabidopsis thaliana showed fertility defect phenotypes， such as short fruit pod and filament， anther deformity， and low pollen activity. The results of germination rate under salt and drought stress showed that the resistance of Mt-lncRNA167 and Mt-miR167c overexpressing A. thaliana plants was better than that of wild type plants. In addition， the hypocotyls of Mt-lncRNA167 and Mt-miR167c overexpressing A. thaliana plants were significantly longer than that of the wild type plants. In conclusion， Mt-lncRNA167 was predicted to be the precursor of Mt-miR167c， while Mt-miR167c inhibited the expression of ARF6/8. The regulatory network of Mt-lncRNA167-Mt-miR167c-ARF6/8 may play an important role in stress tolerance and developmental regulation of M. truncatula.

Key words: alfalfa, long non-coding RNAs, microRNAs, abiotic stress, growth and development

Jia-ju ZHANG, Jie YU, Ming-na LI, Jun-mei KANG, Qing-chuan YANG, Rui-cai LONG. Identification and functional analysis of lncRNA167 and its cleavage product miR167c in Medicago truncatula[J]. Acta Prataculturae Sinica, 2022, 31(1): 164-180.

Figures/Tables 15

References 47

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引物名称Primer	正向引物序列Forward sequence （5′-3′）	反向引物序列Reverse sequence （5′-3′）
Mt-lncRNA167	CAAATTCCAAACCCTAGCCAACT	AAGTTCCTTCCTTCAAAACCAAA
Pre-Mt-miR167c	TCTTTTCCTCAACTCTGCTATAT	CATAATCAACTTTTTTAAGAGCA
qRT-Mt-lncRNA167	GTAGAAGCAGCTAGCATCACT	AACCTACAAAGGAGGCCAGT
qRT-Mt-miR167c	TGAAGCTGCCAGCATGATCTA	试剂盒中反向引物 Reverse primer in kit
qRT-MtARF6	AGAGGGGTGTAGAGTGTAAC	CAAAATAAGCAGAAAATGAC
qRT-MtARF8	ATGATGTGCTTCTCCTTGGC	ATCTTCTGAATATCTTCCGG
qRT-AtARF6	CCTCATGAAGGAGAGAAAAGAGT	AGCAGCAACCTGTTCACTGT
qRT-AtARF8	TGTAGCTGTCTCAGGGTAACT	GGTCTGACCAAACTGAATGTCT
qRT-Mtactin	ACACTGTGCCAATCTATGAGGG	TTGTCCATCAGGAAGTTCATAGTTT
qRT-Atactin	TCCATCGATTGTTCACAGGA	TCACCACCACGAACCAGATA
U6	CGCACAAATCGAGAAATGGTCC	试剂盒中反向引物 Reverse primer in kit
35S	AGAAAATCTTCGTCAACATGGTGGA	\
GUS	TTCCTGATTATTGACCCACACTTTG	\

引物名称Primer	正向引物序列Forward sequence （5′-3′）	反向引物序列Reverse sequence （5′-3′）
Mt-lncRNA167	CAAATTCCAAACCCTAGCCAACT	AAGTTCCTTCCTTCAAAACCAAA
Pre-Mt-miR167c	TCTTTTCCTCAACTCTGCTATAT	CATAATCAACTTTTTTAAGAGCA
qRT-Mt-lncRNA167	GTAGAAGCAGCTAGCATCACT	AACCTACAAAGGAGGCCAGT
qRT-Mt-miR167c	TGAAGCTGCCAGCATGATCTA	试剂盒中反向引物 Reverse primer in kit
qRT-MtARF6	AGAGGGGTGTAGAGTGTAAC	CAAAATAAGCAGAAAATGAC
qRT-MtARF8	ATGATGTGCTTCTCCTTGGC	ATCTTCTGAATATCTTCCGG
qRT-AtARF6	CCTCATGAAGGAGAGAAAAGAGT	AGCAGCAACCTGTTCACTGT
qRT-AtARF8	TGTAGCTGTCTCAGGGTAACT	GGTCTGACCAAACTGAATGTCT
qRT-Mtactin	ACACTGTGCCAATCTATGAGGG	TTGTCCATCAGGAAGTTCATAGTTT
qRT-Atactin	TCCATCGATTGTTCACAGGA	TCACCACCACGAACCAGATA
U6	CGCACAAATCGAGAAATGGTCC	试剂盒中反向引物 Reverse primer in kit
35S	AGAAAATCTTCGTCAACATGGTGGA	\
GUS	TTCCTGATTATTGACCCACACTTTG	\

顺式作用元件Cis-acting element	核心序列 Core sequence	功能 Function
TATA-box	TATATAA/ATTATA/TATA	在转录开始-30左右的核心启动子元件Core promoter element around -30 of transcription start
CAAT-box	CAAT/CAAAT	启动子和增强子区域常见顺式作用元件Common cis-acting element in promoter and enhancer regions
MBS	CAACTG	MYB结合位点，受到干旱胁迫诱导MYB binding site involved in drought-inducibility
TCA-element	CCATCTTTTT	参与水杨酸反应的顺式作用元件Cis-acting element involved in salicylic acid responsiveness
LTR	CCGAAA	参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness
TCCC-motif	TCTCCCT	参与光响应的顺式作用元件Light responsive element
TCT-motif	TCTTAC	参与光响应的顺式作用元件Light responsive element
I-box	GTATAAGGCC	参与光响应的顺式作用元件Light responsive element
Gap-box	CAAATGAA（A/G）A	参与光响应的顺式作用元件Light responsive element
chs-CMA1a	TTACTTAA	参与光响应的顺式作用元件Light responsive element
GA-motif	ATAGATAA	参与光响应的顺式作用元件Light responsive element

顺式作用元件Cis-acting element	核心序列 Core sequence	功能 Function
TATA-box	TATATAA/ATTATA/TATA	在转录开始-30左右的核心启动子元件Core promoter element around -30 of transcription start
CAAT-box	CAAT/CAAAT	启动子和增强子区域常见顺式作用元件Common cis-acting element in promoter and enhancer regions
MBS	CAACTG	MYB结合位点，受到干旱胁迫诱导MYB binding site involved in drought-inducibility
TCA-element	CCATCTTTTT	参与水杨酸反应的顺式作用元件Cis-acting element involved in salicylic acid responsiveness
LTR	CCGAAA	参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness
TCCC-motif	TCTCCCT	参与光响应的顺式作用元件Light responsive element
TCT-motif	TCTTAC	参与光响应的顺式作用元件Light responsive element
I-box	GTATAAGGCC	参与光响应的顺式作用元件Light responsive element
Gap-box	CAAATGAA（A/G）A	参与光响应的顺式作用元件Light responsive element
chs-CMA1a	TTACTTAA	参与光响应的顺式作用元件Light responsive element
GA-motif	ATAGATAA	参与光响应的顺式作用元件Light responsive element

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