蒺藜苜蓿lncRNA167及其剪切产物miR167c的鉴定和功能分析

doi:10.11686/cyxb2020501

摘要/Abstract

摘要：

长链非编码RNA（long non-coding RNA， lncRNA）是一类长度大于200个核苷酸的功能性RNA分子，lncRNA在植物生长发育以及生物和非生物胁迫响应过程中发挥着重要作用。lncRNA常与microRNA（miRNA）关联发挥作用。本研究从蒺藜苜蓿中克隆一个预测的新lncRNA，该lncRNA与蒺藜苜蓿miR167c在染色体上位置重合，命名为Mt-lncRNA167，并推测Mt-lncRNA167作为Mt-miR167c的剪切前体发挥作用。靶基因预测分析结果表明，生长素响应因子6/8（auxin response factor 6/8）是miR167c的两个靶基因。表达模式分析表明，Mt-lncRNA167主要在叶中表达，Mt-lncRNA167和Mt-miR167c对100 mmol·L^-1NaCl、5% PEG 6000和4 ℃低温胁迫处理均有响应，与Mt-lncRNA167启动子顺式作用元件功能预测结果一致。在盐、干旱、低温胁迫条件下，Mt-lncRNA167与Mt-miR167c表达模式基本相同，与靶基因ARF6/8表达模式相反。Mt-lncRNA167与Mt-miR167c超表达转化拟南芥植株表现出生育缺陷的表型，即果荚和花丝显著缩短，花药不能正常释放花粉，花粉活力降低。盐和干旱胁迫下发芽率结果表明Mt-lncRNA167和Mt-miR167c超表达植株抗性优于野生型。此外，Mt-lncRNA167和Mt-miR167c超表达植株下胚轴显著长于野生型。综合以上，推测Mt-lncRNA167作为Mt-miR167c的前体发挥作用，Mt-miR167c抑制ARF6/8基因的表达，Mt-lncRNA167-Mt-miR167c-ARF6/8作用网络在蒺藜苜蓿抗逆和发育调控方面发挥重要作用。

关键词: 苜蓿, 长链非编码RNA, microRNA, 非生物胁迫, 生长发育

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

张家驹, 于洁, 李明娜, 康俊梅, 杨青川, 龙瑞才. 蒺藜苜蓿lncRNA167及其剪切产物miR167c的鉴定和功能分析[J]. 草业学报, 2022, 31(1): 164-180.

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.

图/表 15

表1 本试验所用到的引物序列

Table 1 Primers used in the study

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	\

图1 Mt-lncRNA167与Mt-miR167c的鉴定A： Mt-lncRNA167与Mt-miR167c在染色体上的位置关系 The position relationship between Mt-lncRNA167 and Mt-miR167c on chromosome； B： Mt-miRNA167a/b/c的二级结构 Secondary structure of Mt-miRNA167a/b/c； C： At-miR167a/b/c/d和Mt-miRNA167a/b/c的多序列比对结果Multiple sequence alignment results of At-miR167a/b/c/d and Mt-miRNA167a/b/c.

Fig.1 Identification of Mt-lncRNA167 and Mt-miR167c

图2 miR167成熟体序列碱基保守性分析

Fig.2 Analysis of base conservatism of miR167 mature sequence

图3 pre-miR167s的亲缘关系树

Fig.3 Phylogenetic tree of pre-miR167s

表2 Mt-lncRNA167的启动子顺式作用元件分析

Table 2 Cis-acting element analysis of promoter of Mt-lncRNA167

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

表3 psRNAtarget预测Mt-miR167c的靶基因

Table 3 Prediction of the target gene of Mt-miR167c by psRNAtarget

靶基因符号

Gene symbol

靶基因位置标签

Locus tag

靶基因名称

Gene description

与miRNA互补位点

Complementary sites with miRNA

LOC11413758

MTR_3g064050

生长素响应因子8

Auxin response factor 8

miRNA 21 AUCUAGUACGACCGUCGAAGU 1

Target 1683 UAGAUCAGGCUGGCAGCUUGU 1703

LOC11446854

MTR_2g018690

生长素响应因子6

Auxin response factor 6

miRNA 21 AUCUAGUACGACCGUCGAAGU 1

Target 3316 GAGAUCAGGCUGGCAGCUUGU 3336

LOC11442326

MTR_5g066710

E3泛素蛋白连接酶基因

E3 ubiquitin-protein ligase

miRNA 21 AUCUAGUACGACCGUCGAAGU 1

Target 11475 GAGAUCAGGCUGGCAGCUUGU 11495

LOC11442155

MTR_7g077700

RNA聚合酶Ⅱ转录中介蛋白基因

RNA polymerase Ⅱ transcription mediators

miRNA 21 AUCUAGUACGACCGUCGAAGU 1

Target 250 GAGAUCAGGCUGGCAGCUUGU 270

图4 Mt-lncRNA167、Mt-miR167c、MtARF6/8在不同组织中的表达标准差来自3个生物学重复，不同小写字母代表差异显著（P<0.05），下同。Bars represent the SD of three biological replicates and different lowercase letters indicate significant difference （P<0.05）， the same below.

Fig.4 The relative expression of Mt-lncRNA167， Mt-miR167c， MtARF6/8 in different tissues of M. truncatula

图5 Mt-lncRNA167、Mt-miR167c、MtARF6/8 在盐胁迫（100 mmol·L-1 NaCl）处理下的表达

Fig.5 The relative expression of Mt-lncRNA167， Mt-miR167c， MtARF6/8 under NaCl （100 mmol·L-1） treatment

图6 Mt-lncRNA167、Mt-miR167c、MtARF6/8 在低温胁迫（4 ℃）处理下的表达

Fig.6 The relative expression of Mt-lncRNA167， Mt-miR167c， MtARF6/8 under cold stress （4 ℃） treatment

图7 Mt-lncRNA167、Mt-miR167c、MtARF6/8 在干旱胁迫（5% PEG 6000）处理下的表达

Fig.7 The relative expression of Mt-lncRNA167， Mt-miR167c， MtARF6/8 under drought （5% PEG 6000） treatment

图8 超表达拟南芥中Mt-lncRNA167、Mt-miR167c的表达量分析A：超表达拟南芥的Mt-lncRNA167相对表达量The relative expression level of Mt-lncRNA167 in the overexpressed Arabidopsis； B：超表达拟南芥Mt-miR167c的相对表达量The relative expression level of Mt-miR167c in the overexpressed Arabidopsis； C：超表达Mt-lncRNA167拟南芥中Mt-miR167c、AtARF6、AtARF8的相对表达量The relative expression levels of Mt-miR167c， AtARF6 and AtARF8 in overexpressed Mt-lncRNA167Arabidopsis； D：超表达Mt-miR167c拟南芥中AtARF6、AtARF8的相对表达量The relative expression levels of AtARF6 and AtARF8 in overexpressed Mt-miR167cArabidopsis. WT：野生型Wild type； OE1， 2， 3…：超表达株系Overexpression line 1， 2， 3…；下同The same below.

Fig.8 Analysis of the relative expression level of Mt-lncRNA167 and Mt-miR167c in the overexpressed Arabidopsis

图9 Mt-lncRNA167、Mt-miR167c超表达拟南芥的花结构A： WT与超表达拟南芥的花序结构Inflorescence structure of WT and overexpressed Arabidopsis； B： WT与超表达拟南芥的角果Pod of WT and overexpressed Arabidopsis； C~E： WT与超表达拟南芥的花结构Flower structure of WT and overexpressed Arabidopsis； F~H： WT与超表达拟南芥的雄蕊和雌蕊Stamens and pistil of WT and overexpressed Arabidopsis；图A和B标尺=1 mm Scales in A and B=1 mm；图C~H标尺=0.5 mm Scales in C-H=0.5 mm.

Fig.9 The flower structure of overexpressed Mt-lncRNA167 and Mt-miR167cArabidopsis

图10 WT和超表达拟南芥的幼苗和下胚轴长度A： 1周龄拟南芥幼苗One-week old Arabidopsis seedlings； B： 1周龄拟南芥幼苗下胚轴长度The hypocotyl length of one-week old Arabidopsis seedlings；标尺=1 mm Scale=1 mm.

Fig.10 Seeding and hypocotyl length of WT and overexpressed Arabidopsis

图11 拟南芥花粉的体外萌发A： WT花粉The pollen of WT； B： OE-Mt-lncRNA167植株花粉The pollen of OE-Mt-lncRNA167； C： OE-Mt-miR167c植株花粉The pollen of OE-Mt-miR167c； D： WT、OE-Mt-lncRNA167植株、OE-Mt-miR167c植株花粉萌发率统计Statistics of pollen germination rate of WT， OE-Mt-lncRNA167 and OE-Mt-miR167c plants；标尺Scale=100 μm.

Fig.11 In vitro germination of Arabidopsis pollen

图12 不同浓度盐胁迫和干旱胁迫下转基因拟南芥的发芽率

Fig.12 Germination rate of overexpressed Arabidopsis under different salinity and drought stress

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