蒺藜苜蓿lncRNA167及其剪切产物miR167c的鉴定和功能分析  

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

作　　者：张家驹 于洁 李明娜[1] 康俊梅[1] 杨青川[1] 龙瑞才[1] ZHANG Jia-ju;YU Jie;LI Ming-na;KANG Jun-mei;YANG Qing-chuan;LONG Rui-cai(Institute of Animal Science,the Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《草业学报》2022年第1期164-180,共17页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(32071865)资助。

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MtmiR167c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,MtmiR167c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。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.

关 键 词：苜蓿 长链非编码RNA MICRORNA 非生物胁迫 生长发育 

分 类 号：S541.9[农业科学—作物学]