响应地黄连作障碍LncRNA-RgAGT2的克隆及表达分析
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摘要
连作障碍严重影响地黄的产量和品质,对其分子机制的探寻是地黄研究领域的重要问题之一。该研究基于前期数字表达谱(DGE)分析筛选到的特异响应连作障碍转录本Unigene29334_All,利用hi TAIL-PCR和RACE技术克隆到长度为1 573 bp的完整转录产物。借助ORF Finder预测发现,克隆产物所有ORF均小于300 nt,证实此转录产物为长链非编码RNA(long non-coding RNA,LncRNA)。与地黄转录组数据库比对发现,此LncRNA与丙氨酸-乙醛酸转氨酶2基因(Rg AGT2)部分区域同源,因此命名为LncRNA-RgATG2。为进一步研究LncRNA-RgAGT2的功能,该研究检测了LncRNA-RgAGT2与Rg AGT2基因在头茬和连作地黄生长发育5个关键时期(苗期,拉线期,膨大前期,膨大中期,膨大后期)的表达模式,结果暗示,LncRNA-Rg AGT2可能与地黄Rg AGT2基因存在调控关系。同时,连作地黄LncRNA-RgAGT2表达水平在各个时期均发生不同程度的变化,可以作为地黄连作障碍的"诊断标签"。该研究首次证实LncRNA-RgAGT2参与地黄连作障碍应答及调控过程,是对地黄连作障碍分子机制的有力补充。
The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334_All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.
The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334_All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.
引文
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[15] Wang H,Fang J,Liang C,et al. Computation-assisted SiteFind-ing-PCR for isolating flanking sequence tags in rice[J]. Biotech-niques,2011,51(6):421.
[16] Liu X,Li D,Zhang D,et al. A novel antisense long noncodingRNA,TWISTED LEAF,maintains leaf blade flattening by regula-ting its associated sense R2R3-MYB gene in rice[J]. New Phy-tol,2018,218(2):774.
[17]张重义,陈慧,杨艳会,等.连作对地黄根际土壤细菌群落多样性的影响[J].应用生态学报,2010,21(11):2843.
[18] Zhang B,Li X,Wang F,et al. Assaying the potential autotoxinsand microbial community associated with Rehmannia glutinosa re-plant problems based on its'autotoxic circle'[J]. Plant Soil,2016,407(1/2):307.
[19] Yan H Y,Ming J L,Xin Y L,et al. Transcriptome-wide identifi-cation of the genes responding to replanting disease in Rehmanniaglutinosa L. roots[J]. Mol Biol Rep,2015,42(5):881.
[20] Reik W. Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629.
[21] Chekanova J A. Long non-coding RNAs and their functions inplants[J]. Curr Opin Plant Biol,2015,27:207.
[22] Mathieu E L,Belhocine M,Dao L T,et al. Functions of lncRNA indevelopment and diseases[J]. M S-Med Sci,2014,30(8/9):790.
[23] Zhu Q H,Wang M B. Molecular functions of long non-codingRNAs in plants[J]. Genes,2012,3(1):176.
[24]廖江铨,王阶,刘咏梅,等.高通量测序筛选冠心病血瘀证相关lncRNA-miRNA-mRNA调控网络[J].中国实验方剂学杂志,2017,23(19):28.
[25] Wu Z,Liu X,Liu L,et al. Regulation of lncRNA expression[J].Cell Mol Biol Lett,2014,19(4):561.
[26] Liepman A H,Olsen L J. Alanine aminotransferase homologs cata-lyze the glutamate:glyoxylate aminotransferase reaction in peroxi-somes of Arabidopsis[J]. Plant Physiol,2003,131(1):215.
[27] Luo B,Norris C,Bolstad E S D,et al. Protein quaternary structureand expression levels contribute to peroxisomal targeting sequence1-mediated peroxisomal import of human soluble epoxide hydro-lase[J]. J Mol Biol,2008,380(1):31.
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[29] Kao Y T,Gonzalez K L,Bartel B. Peroxisome function,biogenesis,and dynamics in plants[J]. Plant Physiol,2017,176(1):162.
[30] Banjoko A,Trelease R N. Development and application of an in vivoplant peroxisome import system[J]. Plant Physiol,1995,107(4):1201.
[31] Toufighi K,Brady S M,Austin R,et al. The botany array re-source:e-northerns,expression angling,and promoter analyses[J]. Plant J,2005,43(1):153.
[32] Nunes-Nesi A,Florian A,Howden A,et al. Is there a metabolicrequirement for photorespiratory enzyme activities in heterotrophictissues?[J]. Mol Plant,2014,7(1):248.
[33] Jacoby R P,Harvey M A,Taylor N L. Application of selected re-action monitoring mass spectrometry to field-grown crop plants toallow dissection of the molecular mechanisms of abiotic stress tol-erance[J]. Front Plant Sci,2013,4(1):20.
[34] Taler D,Galperin M,Benjamin I,et al. Plant eR genes that en-code photorespiratory enzymes confer resistance against disease[J]. Plant Cell,2004,16(1):172.
[35] Wang H,Chung P J,Liu J,et al. Genome-wide identification oflong noncoding natural antisense transcripts and their responses tolight in Arabidopsis[J]. Genome Res,2014,24(3):444.
[36] Seo J S,Sun H X,Park B S,et al. Elf18-induced long noncoding RNAassociates with mediator to enhance expression of innate immune re-sponse genes in Arabidopsis[J]. Plant Cell,2017,29(5):1024.
[37] Kim D H,Xi Y,Sung S. Modular function of long noncodingRNA,COLDAIR,in the vernalization response[J]. PLoS Genet,2017,13(7):e1006939.
[38] Wang M,Wu H J,Fang J,et al. A long noncoding RNA involvedin rice reproductive development by negatively regulating osa-miR160[J]. Sci Bull,2017,62(7):470.
[2]张重义,林文雄.药用植物的化感自毒作用与连作障碍[J].中国生态农业学报,2009,17(1):189.
[3]杜家方,尹文佳,李娟,等.连作地黄根际土壤中酚酸类物质的动态变化[J].中国中药杂志,2009,34(8):948.
[4]杜家方,尹文佳,张重义,等.不同间隔年限地黄土壤的自毒作用和酚酸类物质含量[J].生态学杂志,2009,28(3):445.
[5] Chon S U,Choi S K,Jung S,et al. Effects of alfalfa leaf extractsand phenolic allelochemicals on early seedling growth and rootmorphology of alfalfa and barnyard grass[J]. Crop Prot,2002,21(10):1077.
[6] Yu J Q,Ye S F,Zhang M F,et al. Effects of root exudatesandaqueous root extracts of cucumber(Cucumis sativus)andallelo-chemicals on photosynthesis and antioxidant enzymes incucumber[J]. Biochem Syst Ecol,2003,31(2):129.
[7] Okazaki Y,Furuno M,Kasukawa T,et al. Analysis of the mousetranscriptome based on functional annotation of 60,770 full-length c DNAs[J]. Nature,2002,420:563.
[8] Li W,Li C,Li S,et al. Long noncoding RNAs that respond toFusarium oxysporum infection in'Cavendish'banana(Musa acu-minata)[J]. Sci Rep-UK,2017,7(1):16939.
[9] Laurent S,Georges,Wahlestedt,et al. The Landscape of long non-coding RNA classification[J]. Trends Genet,2015,31(5):239.
[10] Cech T R,Steitz J A. The noncoding RNA revolution-trashing oldrules to forge new ones[J]. Cell,2014,157(1):77.
[11] Rinn J L,Chang H Y. Genome regulation by long noncodingRNAs[J]. Annu Rev Biochem,2012,81(1):145.
[12] Singh U,Khemka N,Rajkumar M S,et al. PLncPRO for predic-tion of long non-coding RNAs(lncRNAs)in plants and its ap-plication for discovery of abiotic stress-responsive lncRNAs in riceand chickpea[J]. Nucleic Acids Res,2017,45(22):e183.
[13] Golicz A A,Bhalla P L,Singh M B. lncRNAs in plant and animalsexual reproduction[J]. Trends Plant Sci,2018,23(3):195.
[14] Liu Y G,Chen Y. High-efficiency thermal asymmetric interlacedPCR for amplification of unknown flanking sequences[J]. Bio-techniques,2007,43(5):649.
[15] Wang H,Fang J,Liang C,et al. Computation-assisted SiteFind-ing-PCR for isolating flanking sequence tags in rice[J]. Biotech-niques,2011,51(6):421.
[16] Liu X,Li D,Zhang D,et al. A novel antisense long noncodingRNA,TWISTED LEAF,maintains leaf blade flattening by regula-ting its associated sense R2R3-MYB gene in rice[J]. New Phy-tol,2018,218(2):774.
[17]张重义,陈慧,杨艳会,等.连作对地黄根际土壤细菌群落多样性的影响[J].应用生态学报,2010,21(11):2843.
[18] Zhang B,Li X,Wang F,et al. Assaying the potential autotoxinsand microbial community associated with Rehmannia glutinosa re-plant problems based on its'autotoxic circle'[J]. Plant Soil,2016,407(1/2):307.
[19] Yan H Y,Ming J L,Xin Y L,et al. Transcriptome-wide identifi-cation of the genes responding to replanting disease in Rehmanniaglutinosa L. roots[J]. Mol Biol Rep,2015,42(5):881.
[20] Reik W. Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629.
[21] Chekanova J A. Long non-coding RNAs and their functions inplants[J]. Curr Opin Plant Biol,2015,27:207.
[22] Mathieu E L,Belhocine M,Dao L T,et al. Functions of lncRNA indevelopment and diseases[J]. M S-Med Sci,2014,30(8/9):790.
[23] Zhu Q H,Wang M B. Molecular functions of long non-codingRNAs in plants[J]. Genes,2012,3(1):176.
[24]廖江铨,王阶,刘咏梅,等.高通量测序筛选冠心病血瘀证相关lncRNA-miRNA-mRNA调控网络[J].中国实验方剂学杂志,2017,23(19):28.
[25] Wu Z,Liu X,Liu L,et al. Regulation of lncRNA expression[J].Cell Mol Biol Lett,2014,19(4):561.
[26] Liepman A H,Olsen L J. Alanine aminotransferase homologs cata-lyze the glutamate:glyoxylate aminotransferase reaction in peroxi-somes of Arabidopsis[J]. Plant Physiol,2003,131(1):215.
[27] Luo B,Norris C,Bolstad E S D,et al. Protein quaternary structureand expression levels contribute to peroxisomal targeting sequence1-mediated peroxisomal import of human soluble epoxide hydro-lase[J]. J Mol Biol,2008,380(1):31.
[28] Corpas F J. Peroxisomes as a source of reactive oxygen speciesand nitric oxide signal molecules in plant cells[J]. Trends PlantSci,2001,6(4):145.
[29] Kao Y T,Gonzalez K L,Bartel B. Peroxisome function,biogenesis,and dynamics in plants[J]. Plant Physiol,2017,176(1):162.
[30] Banjoko A,Trelease R N. Development and application of an in vivoplant peroxisome import system[J]. Plant Physiol,1995,107(4):1201.
[31] Toufighi K,Brady S M,Austin R,et al. The botany array re-source:e-northerns,expression angling,and promoter analyses[J]. Plant J,2005,43(1):153.
[32] Nunes-Nesi A,Florian A,Howden A,et al. Is there a metabolicrequirement for photorespiratory enzyme activities in heterotrophictissues?[J]. Mol Plant,2014,7(1):248.
[33] Jacoby R P,Harvey M A,Taylor N L. Application of selected re-action monitoring mass spectrometry to field-grown crop plants toallow dissection of the molecular mechanisms of abiotic stress tol-erance[J]. Front Plant Sci,2013,4(1):20.
[34] Taler D,Galperin M,Benjamin I,et al. Plant eR genes that en-code photorespiratory enzymes confer resistance against disease[J]. Plant Cell,2004,16(1):172.
[35] Wang H,Chung P J,Liu J,et al. Genome-wide identification oflong noncoding natural antisense transcripts and their responses tolight in Arabidopsis[J]. Genome Res,2014,24(3):444.
[36] Seo J S,Sun H X,Park B S,et al. Elf18-induced long noncoding RNAassociates with mediator to enhance expression of innate immune re-sponse genes in Arabidopsis[J]. Plant Cell,2017,29(5):1024.
[37] Kim D H,Xi Y,Sung S. Modular function of long noncodingRNA,COLDAIR,in the vernalization response[J]. PLoS Genet,2017,13(7):e1006939.
[38] Wang M,Wu H J,Fang J,et al. A long noncoding RNA involvedin rice reproductive development by negatively regulating osa-miR160[J]. Sci Bull,2017,62(7):470.