利用CRISPR技术鉴定拟南芥miR171a的功能
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摘要
miRNA是一段长约21 nt的RNA小分子,能够影响多种植物生理生化功能。CRISPR/Cas9是近年发展的可以靶向编辑DNA序列的技术,已广泛应用于多种植物。miR171a是一类保守的miRNA家族,参与对植物生长发育和逆境胁迫的调控。利用CRISPR/Cas9技术鉴定拟南芥miR171a的功能,结果表明基因编辑拟南芥株系中的miR171a表达量降低,靶基因SCL22表达量上调。干旱处理后基因编辑拟南芥株系中的脯氨酸含量上升幅度小于野生型。基因编辑拟南芥株系的叶绿素含量有不同程度的降低,株高和生长势增强。干旱胁迫结果显示对miR171的抑制使得拟南芥抗旱能力减弱,表明利用CRISPR技术鉴定miRNA的功能是有效的。
microRNA( miRNA) is a small RNA molecule of about 21 nt that can affect the physiological and biochemical functions of various plants. CRISPR/Cas9 is a well-developed system for edting target DNA sequences and has been successfully applied in many plants. miR171 a is a kind of conserved miRNA family,which is involved in the regulation of plant growth and development and stress under stress. In this study,CRISPR/Cas9 technique was used to identify the functions of miR171 a in Arabidopsis thaliana. The results showed that the expression of miR171 a decreased in gene edited Arabidopsis lines. The expression of target gene SCL22 was up regulated. The increase of proline content in Arabidopsis lines was smaller than that of wild type after drought stress. The chlorophyll content,plant height and growth potential of gene edited Arabidopsis lines decreased. Drought stress results showed that inhibition of miR171 weakened the drought resistance of Arabidopsis lines. These results showed that CRISPR technology was effective in identifying the functions of miRNA.
microRNA( miRNA) is a small RNA molecule of about 21 nt that can affect the physiological and biochemical functions of various plants. CRISPR/Cas9 is a well-developed system for edting target DNA sequences and has been successfully applied in many plants. miR171 a is a kind of conserved miRNA family,which is involved in the regulation of plant growth and development and stress under stress. In this study,CRISPR/Cas9 technique was used to identify the functions of miR171 a in Arabidopsis thaliana. The results showed that the expression of miR171 a decreased in gene edited Arabidopsis lines. The expression of target gene SCL22 was up regulated. The increase of proline content in Arabidopsis lines was smaller than that of wild type after drought stress. The chlorophyll content,plant height and growth potential of gene edited Arabidopsis lines decreased. Drought stress results showed that inhibition of miR171 weakened the drought resistance of Arabidopsis lines. These results showed that CRISPR technology was effective in identifying the functions of miRNA.
引文
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[2]Cesar L,Kasschau K D,Rector M A,et al..Endogenous and silencing-associated small RNAs in plants[J].Plant Cell,2002,14(7):1605-19.
[3]Mette M F,van der Winden J,Matzke M,et al..Short RNAs can identify new candidate transposable element families in Arabidopsis[J].Plant Physiol.,2002,130(1):6-9.
[4]Park W,Li J,Song R,et al..CARPEL FACTORY,a dicer homolog,and HEN1,a novel protein,act in microRNAmetabolism in Arabidopsis thaliana[J].Curr.Biol.,2002,12(17):1484-1495.
[5]Bohmert K,Camus I,Bellini C,et al..AGO1 defines a novel locus of Arabidopsis controlling leaf development[J].EMBOJ.,1998,17(1):170-180.
[6]Vaucheret H.Plant ARGONAUTES[J].Trends Sci.,2008,13(7):350-358.
[7]Hendelman A,Buxdorf K,Stav R,et al..Inhibition of lamina outgrowth following Solanum lycopersicum AUXIN RESPONSEFACTOR 10(SlARF10)derepression[J].Plant Mol.Biol.,2012,78(6):561-576.
[8]Han Y,Zhang X,Wang Y,et al..The suppression of WRKY44 by GIGANTEA-miR172 pathway is involved in drought response of Arabidopsis thaliana[J].PLoS ONE,2013,8(11):e73541.
[9]Zhou Y,Gao F,Liu R,et al..De novo sequencing and analysis of root transcriptome using 454 pyrosequencing to discover putative genes associated with drought tolerance in Ammopiptanthus mongolicus[J].BMC Genomics,2012,13(1):266.
[10]何炫程,吕鹤书,黄捷飞,等.植物miRNA靶基因验证研究进展[J].生物技术进展,2017,7(2):102-105.He X C,Lv H S,Huang J F,et al..Progress in plant miRNAtarget gene verification[J].Adv.Biotechnol.,2017,7(2):102-105.
[11]Franco-Zorrilla J M,Valli A,Todesco M,et al..Target mimicry provides a new mechanism for regulation of microRNAactivity[J].Nat.Genetics,2007,39(8):1033-1037.
[12]Yan J,Gu Y,Jia X,et al..Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis[J].Plant Cell,2012,24(2):415-427.
[13]Tang G,Yan J,Gu Y,et al..Construction of short tandem target mimic(STTM)to block the functions of plant and animal microRNAs[J].Methods,2012,58(2):118-125.
[14]Voytas D F.Plant genome engineering with sequence-specific nucleases[J].Annu.Rev.Plant Biol.,2013,64(64):327-350.
[15]Li J F,Norville J E,Aach J,et al..Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nat.Biotechnol.,2013,31(8):688-691.
[16]Shan Q,Wang Y,Li J,et al..Targeted genome modification of crop plants using a CRISPR-Cas system[J].Nat.Biotechnol.,2013,31(8):686-688.
[17]Sun X,Hu Z,Chen R,et al..Targeted mutagenesis in soybean using the CRISPR-Cas9 system[J].Sci.Rep.,2015,5:10342.
[18]Brooks C,Nekrasov V,Lippman Z,et al..Efficient gene editing in tomato in the first generation using the CRISPR/Cas9system[J].Plant Physiol.,2014,88:204-217.
[19]Zhu X,Leng X,Sun X,et al..Discovery of conservation and diversification of genes by phylogenetic analysis based on global genomes[J].Plant Genome,2015,doi:10.3835/plantgenome2014.10.0076.
[20]张静文.普通野生稻(Oryza rufipogon Griff.)干旱胁迫相关microRNAs的鉴定与功能分析[D].北京:中国农业大学,博士学位论文,2017.Zhang J W.Identification and functional analysis of droughtrelated microRNAs in common wild rice(Oryza rufipogon Griff.)[D].Beijing:China Agricultural University,Doctor Dissertation,2017.
[21]Zhou J,Deng K,Cheng Y,et al..CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice[J].Front.Plant Sci.,2017,8:1598.
[22]Zhao Y,Zhang C,Liu W,et al..An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9design[J].Sci.Rep.,2016,6:23890.
[23]Mao Y,Zhang H,Xu N,et al..Application of the CRISPR-Cas system for efficient genome engineering in plants[J].Mol.Plant,2013,6(6):2008-2011.
[24]Feng Z,Zhang B,Ding W,et al..Efficient genome editing in plants using a CRISPR/Cas system[J].Cell Res.,2013,23(10):1229-1232.