观赏海棠中McmiR160a的克隆及调控红叶着色的功能分析
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摘要
【目的】为了验证观赏海棠叶片McmiR160a对花色素苷代谢的调控作用。【方法】以观赏海棠常色红叶品种‘王族’为试验材料,确定miR160a的前体基因序列并对其靶基因进行预测,克隆McmiR160a前体序列并构建过表达载体并瞬时转入‘王族’组培苗。通过高效液相色谱和qRT-PCR分析瞬时侵染植株中花色素苷的含量及其合成途径中结构基因的表达量。【结果】结果表明,在观赏海棠中过表达McmiR160a可以降低花色素苷的含量,同时降低其靶基因和花色素苷代谢途径关键基因表达水平。【结论】研究结果表明,观赏海棠中McmiR160a对花色素苷的合成具有负调控作用。
【Objective】This experiment was to verify the regulation of miR160a on anthocyanin biosynthesis in Malus spp.leaves.【Methods】We used the leaves of ever-red leaf cultivar ‘Royalty’ of Malus spp.as experimental material.The sequence of miR160a precursor gene and its target genes were predicted.The precursor sequence of McmiR160a was cloned and was transiently over-expressed into the‘Royalty'tissue culture seedlings.And then,the contents of anthocyanin and the expressions of key genes in the pathway of anthocyanin biosynthesis were analyzed by HPLC and qRT-PCR.【Results】The results showed that over-expressing of McmiR160a in Malus spp.leaves decreased the expression of its target gene,the anthocyanin contents,and the expressions of key genes in anthocyanin biosynthesis pathway.【Conclusion】The results suggested that McmiR160a negatively regulated anthocyanins biosynthesis Malus spp..
【Objective】This experiment was to verify the regulation of miR160a on anthocyanin biosynthesis in Malus spp.leaves.【Methods】We used the leaves of ever-red leaf cultivar ‘Royalty’ of Malus spp.as experimental material.The sequence of miR160a precursor gene and its target genes were predicted.The precursor sequence of McmiR160a was cloned and was transiently over-expressed into the‘Royalty'tissue culture seedlings.And then,the contents of anthocyanin and the expressions of key genes in the pathway of anthocyanin biosynthesis were analyzed by HPLC and qRT-PCR.【Results】The results showed that over-expressing of McmiR160a in Malus spp.leaves decreased the expression of its target gene,the anthocyanin contents,and the expressions of key genes in anthocyanin biosynthesis pathway.【Conclusion】The results suggested that McmiR160a negatively regulated anthocyanins biosynthesis Malus spp..
引文
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[13]Boer DR,Freire-Rios A,van den Berg WA,Saaki T,Manfield IW,Kepinski S,López-Vidrieo I,Franco-Zorrilla JM,de Vries SC,Solano R,Weijers D,Coll M.Structural basis for DNA binding specificity by the auxin-dependent ARFtranscription factors[J].Cell,2014,156(3):577-589
[14]LigangR,GuiliangT.Identification of sucrose-responsive microRNAs reveals sucrose-regulated copper accumulations in an SPL7-dependent and independent manner in Arabidopsis thaliana[J].Plant Science,2012,187:59-68
[15]Lei KJ,Lin YM,An GY.miR156modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis[J].Journal of Plant Research,2016,129(2):275-284
[16]Damodharan S,Zhao D,Arazi T.A common miRNA160-based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato[J].the Plant Journal.2016,86(6):458-471
[17]Zhong L,MingZhu S,DeYu X.Regulation of anthocyanin biosynthesis in Arabidopsis thaliana red pap1-D cells metabolically programmed by auxins[J].Planta,2014,239(4):765-781
[2]黄爱优.三角褐指藻miRNA筛选作用机制及代谢流量分析[D].北京:中国科学院大学,2013
[3]陈雪姣.调控miRNA的活性小分子的发现和作用机制研究[D].南京:南京大学,2013
[4]Lin Y,Lai Zhx,Tian Q,Lin Lx,Lai R,Yang Mm,Zhang Dm,Chen Yk and Zhang Zh.Endogenous target mimics down-regulate miR160 mediation of ARF10,-16,and-17cleavage during somatic embryogenesis in Dimocarpus longan Lour[J].Frontiers in plant science,2015,6:956
[5]Pashkovskiy PP,Kartashov AV,Zlobin IE,Pogosyan SI,Kuznetsov VV.Blue light alters miR167expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants[J].Plant physiology and biochemistry,2016,104:146-154
[6]Bai B,Bian H,Zeng Z,Hou N,Shi B,Wang J,Zhu M,Han N.miR393-mediated auxin signaling regulation is involved in root elongation inhibition in response to toxic aluminum stress in Barley[J].plant and cell physiology.2017,58(3):426-439
[7]Xia R,Zhu H,An YQ,Beers EP,Liu Z.Apple miRNAs and tasiRNAs with novel regulatory networks[J].Genome biology,2012,13(6):R47
[8]Morea EG,Valente GT,Nogueira FT.Functional and evolutionary analyses of the miR156and miR529families in land plants[J].BMC Plant Biology,2016,16(1):40
[9]Liu R,Lai B,Hu B,Qin Yh,Hu1Gb,Zhao Jt.Identification of microRNAs and their target genes related to the accumulation of anthocyanins in Litchi chinensis by highthroughput sequencing and degradome analysis[J].Frontiers in plant science,2017,7:2059
[10]Jian H,Zhiyong L,Dazhong Zh.Deregulation of the OsmiR160Target Gene OsARF18Causes Growth and Developmental Defects with an Alteration of Auxin Signaling in Rice[J].Scisence Report,2016,6:29938
[11]Ulmasov T,Hagen G,Guilfoyle T J.Activation and repression of transcription by auxin-response factors[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(10):5844-5849
[12]Wang S,Tiwari S B,Hagen G,Guilfoyle T J.AUXIN RE-SPONSE FACTOR7restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts[J].Plant Cell,2005,17(7):1979-1993
[13]Boer DR,Freire-Rios A,van den Berg WA,Saaki T,Manfield IW,Kepinski S,López-Vidrieo I,Franco-Zorrilla JM,de Vries SC,Solano R,Weijers D,Coll M.Structural basis for DNA binding specificity by the auxin-dependent ARFtranscription factors[J].Cell,2014,156(3):577-589
[14]LigangR,GuiliangT.Identification of sucrose-responsive microRNAs reveals sucrose-regulated copper accumulations in an SPL7-dependent and independent manner in Arabidopsis thaliana[J].Plant Science,2012,187:59-68
[15]Lei KJ,Lin YM,An GY.miR156modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis[J].Journal of Plant Research,2016,129(2):275-284
[16]Damodharan S,Zhao D,Arazi T.A common miRNA160-based mechanism regulates ovary patterning,floral organ abscission and lamina outgrowth in tomato[J].the Plant Journal.2016,86(6):458-471
[17]Zhong L,MingZhu S,DeYu X.Regulation of anthocyanin biosynthesis in Arabidopsis thaliana red pap1-D cells metabolically programmed by auxins[J].Planta,2014,239(4):765-781