蓝莓Vco-miR＿n10的克隆及对干旱的响应分析
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摘要
【目的】MicroRNA(miRNA)是一类内源单链非编码的小分子RNA,在动植物中参与转录后基因表达调控。Vco-miR_n10是蓝莓中的新型miRNA,目前尚未见该miRNA功能方面的报道。本研究对蓝莓VcomiR_n10进行了初步功能分析。【方法】基于前期蓝莓小RNA高通量测序结果,对鉴定到的蓝莓新型miRNA(Vco-miR_n10)的前体序列(224 nt)进行克隆与测序,并利用mfold在线软件对其前体序列的二级结构进行预测。采用Gateway系统构建了35Spro:: pre-Vco-mi R_n10表达载体并转入野生型拟南芥中,对其正常生长及300mmol/L甘露醇处理条件下的表型进行观察与分析。【结果】克隆获得了Vco-mi R_n10的前体序列,该序列能够折叠形成较完美的茎环结构且仅有2个错配碱基对。Vco-miR_n10的成熟体序列位于3'茎臂上。观察正常生长条件下的转基因植株并无显著的表型变化。而在300 mmol/L甘露醇模拟的干旱条件下,Vco-miR_n10-24与VcomiR_n10-52的发芽率(33.53%、37.69%)和绿苗率均显著低于野生型(发芽率67.72%、绿苗率66.33%)。【结论】蓝莓Vco-miR_n10能够响应甘露醇模拟的干旱胁迫,并降低拟南芥对干旱的耐受能力,影响发芽率和绿苗率,说明蓝莓Vco-miR_n10可能在植物响应干旱胁迫过程中发挥一定的调控功能。
【Objective】MicroRNAs(miRNAs) are a class of endogenous, single-stranded and non-coding small RNA molecules that are involved in the regulation of gene expression after transcription in plants and animals. Vco-miR_n10 is a novel miRNA in blueberry, and its function remains unknown. In the present study, a preliminary functional analysis of blueberry Vco-miR_n10 was carried out.【Method】Based on the previous data from high throughput sequencing of small RNA in blueberry, the precursor sequence(224 nt) of the novel miRNA(Vco-miR_n10) was cloned and sequenced.The secondary structure of its precursor sequence was predicted by mfold web server. The expression vector, 35 Spro::preVco-miR_n10, was constructed by Gateway system and transferred into wild type Arabidopsis thaliana and its phenotype under the conditions of normal growth and 300 mmol/L mannitol treatment was observed and analyzed, respectively.【Result】The precursor sequence of Vco-miR_n10 was obtained by cloning, and structure analysis indicated that the precursor shows near-perfect stem-loop structure with no more than 2 mismatches. In addition, the mature sequence was located on the 3'stem arm. No significant phenotypic variation was observed in transgenic plants under the normal growth condition. However, under the drought condition simulated by 300 mmol/L mannitol, the germination rate(33.53%, 37.69%) and cotyledon greening rate(33.53%, 37.69%) of two transgenic lines(Vco-miR_n10-24 and Vco-miR_n10-52) were significantly lower than those of wild type A. thalian(germination rate: 67.72%; cotyledon greening rate:66.33%).【Conclusion】These results indicated that blueberry Vco-miR_n10 could respond to the mannitol-simulated drought stress, and reduce the drought tolerance in A. thalian through affecting seed germination and cotyledon greening, which showed that blueberry Vco-miR_n10 might play a regulatory role in plant response to drought stress.
【Objective】MicroRNAs(miRNAs) are a class of endogenous, single-stranded and non-coding small RNA molecules that are involved in the regulation of gene expression after transcription in plants and animals. Vco-miR_n10 is a novel miRNA in blueberry, and its function remains unknown. In the present study, a preliminary functional analysis of blueberry Vco-miR_n10 was carried out.【Method】Based on the previous data from high throughput sequencing of small RNA in blueberry, the precursor sequence(224 nt) of the novel miRNA(Vco-miR_n10) was cloned and sequenced.The secondary structure of its precursor sequence was predicted by mfold web server. The expression vector, 35 Spro::preVco-miR_n10, was constructed by Gateway system and transferred into wild type Arabidopsis thaliana and its phenotype under the conditions of normal growth and 300 mmol/L mannitol treatment was observed and analyzed, respectively.【Result】The precursor sequence of Vco-miR_n10 was obtained by cloning, and structure analysis indicated that the precursor shows near-perfect stem-loop structure with no more than 2 mismatches. In addition, the mature sequence was located on the 3'stem arm. No significant phenotypic variation was observed in transgenic plants under the normal growth condition. However, under the drought condition simulated by 300 mmol/L mannitol, the germination rate(33.53%, 37.69%) and cotyledon greening rate(33.53%, 37.69%) of two transgenic lines(Vco-miR_n10-24 and Vco-miR_n10-52) were significantly lower than those of wild type A. thalian(germination rate: 67.72%; cotyledon greening rate:66.33%).【Conclusion】These results indicated that blueberry Vco-miR_n10 could respond to the mannitol-simulated drought stress, and reduce the drought tolerance in A. thalian through affecting seed germination and cotyledon greening, which showed that blueberry Vco-miR_n10 might play a regulatory role in plant response to drought stress.
引文
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[13]HAJYZADEH M,TURKTAS M,KHAWAR K M,UNVER T.miR408overexpression causes increased drought tolerance in chickpea[J].Gene,2015,555(2):186-193.doi:10.1016/j.gene.2014.11.002.
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[15]LI W B,WANG T,ZHAN Y H,LI Y G.Overexpression of soybean miR172c confers water deficit and salt tolerance but ABA sensitivity in transgenic Arabidopsis thaliana[J].Journal of Experimental Botany,2015,67(1):175-194.doi:10.1093/jxb/erv450.
[16]ESMAEILI F,SHIRAN B,FALLAHI H,MIRAKHORLI N,BUDAKH,MATINEZ-GOMEZ P.In silico search and biological validation of microRNAs related to drought response in peach and almond[J].Functional&Integrative Genomics,2016,17(2-3):1-13.doi:10.1007/s10142-016-0488-x.
[17]吴美婷,杨晓玉,罗淋淋,莫蓓莘,刘琳.植物microRNA响应非生物胁迫研究进展[J].广东农业科学,2018,45(3):69-80.doi:10.16768/j.issn.1004-874X.2018.03.012.WU M T,YANG X Y,LUO L L,MO B S,LIU L.Research progress of microRNA response to abiotic stress[J].Guangdong Agricultural Sciences,2018,45(3):69-80.doi:10.16768/j.issn.1004-874X.2018.03.012.
[18]郝雨帆,王俊杰,杨丹丹,张静,闫鑫甜,梁卫红.miRNA对水稻非生物胁迫的应答[J].广东农业科学,2014,41(19):5-9.doi:10.3969/j.issn.1004-874X.2014.19.002.HAO Y F,WANG J J,YANG D D,ZHANG J,YAN X T,LIANG WH.Response of miRNA to abiotic stress in Rice(Oryza sativa L.)[J].Guangdong Agricultural Sciences,2014,41(19):5-9.doi:10.3969/j.issn.1004-874X.2014.19.002.
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[20]谷彩红,陈家红,张荃.miRNA参与植物耐逆性调控的研究进展[J].安徽农业科学,2017,45(34):148-151.doi:10.13989/j.cnki.0517-6611.2017.34.047.GU C H,CHEN J H,ZHANG Q.Research progress of miRNAinvolved in the regulation of plant stress tolerance[J].Journal of Anhui Agricultural Sciences,2017,45(34):148-151.doi:10.13989/j.cnki.0517-6611.2017.34.047.
[21]ZHANG B H.MicroRNA:a new target for improving plant tolerance to abiotic stress[J].Journal of Experimental Botany,2015,66(7):1749-1761.doi:10.1093/jxb/erv013.
[22]GHORECHA V,ZHENG Y,LIU L,SUNKAR R,KRISHNAYYA N S R.MicroRNA dynamics in a wild and cultivated species of Convolvulaceae exposed to drought stress[J].Physiology&Molecular Biology of Plants,2017,23(2):291-300.doi:10.1007/s12298-017-0426-y.
[23]肖敏敏,陈信波.microRNA响应植物非生物胁迫的研究进展[J].分子植物育种,2018,16(10):68-73.doi:10.13271/j.mpb.016.003154.XIAO M M,CHEN X B.Advances in microRNA response to abiotic stress in plants[J].Molecular Plant Breeding,2018,16(10):68-73.doi:10.13271/j.mpb.016.003154.
[24]LI W X,OONO Y,ZHU J H,HE X J,WU J M,LIDA K,LU X Y,CUIX P,JIN H L,ZHU J K.The Arabidopsis NFYA5 transcription factor is regulated transcriptionally and posttranscriptionally to promote drought resistance[J].The Plant Cell Online,2008,20(8):2238-2251.doi:10.1105/tpc.108.059444.
[25]HWANG E W,SHIN S J,YU B K,BYUN M O,KWON H B.miR171family members are involved in drought response in Solanum tuberosum[J].Journal of Plant Biology,2011,54(1):43-48.doi:10.1007/s12374-010-9141-8.
[26]FERDOUS J,HUSSAIN S S,SHI B J.Role of microRNAs in plant drought tolerance[J].Plant Biotechnology Journal,2015,13(3):293-305.doi:10.1111/pbi.12318.
[27]罗书芳,崔浪军,王健,屈生宪.干旱胁迫下15种丹参miRNAs差异表达分析[J].广东农业科学,2013,40(5):134-137.doi:10.3969/j.issn.1004-874X.2013.05.040.LUO S F,CUI L J,WANG J,QU S X.Differential expression of miRNAs in 15 species of Salvia miltiorrhiza Bunge under drought stress[J].Guangdong Agricultural Sciences,2013,40(5):134-137.doi:10.3969/j.issn.1004-874X.2013.05.040.
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