梅PmmiR319a与靶基因PmTCP2的验证与表达分析
|
摘要
为验证PmmiR319a与PmTCP2基因的靶标关系,并研究PmmiR319a在梅花芽发育过程中的作用,本研究以梅品种‘大嵌蒂’为试验材料,克隆梅PmmiR319a前体序列及其靶基因PmTCP2,采用5'RACE技术对两者之间的靶标关系进行验证,并进一步分析在花芽发育过程中的表达模式。结果表明,梅PmmiR319a前体序列含有茎环结构,通过序列比对和系统发育分析发现,不同物种的miR319a前体序列的茎环发卡结构和成熟体区域表现出较高的保守性。梅PmTCP2基因全长1 500 bp,编码499个氨基酸。荧光定量PCR分析结果表明,PmmiR319a在梅花发育的前期表达量高,而PmTCP2在梅花发育的中期和后期表达量较高,PmmiR319a与预测靶基因PmTCP2的表达模式呈负相关,表明二者存在负调控的关系,与预测的靶标关系一致。RLM-5'RACE技术验证结果表明,PmmiR319a对其靶基因PmTCP2的mRNA进行了切割,切割位点位于第10和第11个碱基之间。由此可知,PmmiR319a通过调控靶基因PmTCP2影响花的发育。本研究结果为阐明梅花中miR319a的功能提供了一定的理论依据。
In order to verify the relationship between PmmiR319 a and its predicted target gene PmTCP2,and study the role of PmmiR319 a in flower development. The Prunus mume cultivar Daqiandi was used as the experimental material to isolate the Pmm TR319 a precursor sequence and the predicted target gene PmTCP2,and their expression during the flower bud development were analyzed. The relationship between PmmiR319 a and its target gene PmTCP2 was verified with RLM-5'RACE. The results indicated that the PmmiR319 a precursor sequence contained stem and loop structure and the sequence alignment and phylogenetic analysis of PmmiR319 a with miR319 a of other species showed that the sequences of miR319 a precursor were different,and the mature sequences of miR319 a were very conservative. The PmTCP2 gene was 1 500 bp in length and encodes a protein of 499 amino acids. Real-time PCR analysis indicated that PmmiR319 a was highly expressed in the early stage of flower bud development in Prunus mume,while PmTCP2 was higher in the middle and late stages,and the expression of PmmiR319 a was negatively correlated with the expression of the predicted target gene PmTCP2 expression. PmmiR319 a guided-cleavage and target site for the putative PmTCP2 mRNA was validated using RLM-5' RACE. The cleavage site was between the 10 thand 11 thbase of its interaction region. These results indicated that PmmiR319 a plays a role in flower bud development by regulating the target gene PmTCP2. The results of study provide a theoretical basis for the function of miR319 a to the development of Prumes mume
In order to verify the relationship between PmmiR319 a and its predicted target gene PmTCP2,and study the role of PmmiR319 a in flower development. The Prunus mume cultivar Daqiandi was used as the experimental material to isolate the Pmm TR319 a precursor sequence and the predicted target gene PmTCP2,and their expression during the flower bud development were analyzed. The relationship between PmmiR319 a and its target gene PmTCP2 was verified with RLM-5'RACE. The results indicated that the PmmiR319 a precursor sequence contained stem and loop structure and the sequence alignment and phylogenetic analysis of PmmiR319 a with miR319 a of other species showed that the sequences of miR319 a precursor were different,and the mature sequences of miR319 a were very conservative. The PmTCP2 gene was 1 500 bp in length and encodes a protein of 499 amino acids. Real-time PCR analysis indicated that PmmiR319 a was highly expressed in the early stage of flower bud development in Prunus mume,while PmTCP2 was higher in the middle and late stages,and the expression of PmmiR319 a was negatively correlated with the expression of the predicted target gene PmTCP2 expression. PmmiR319 a guided-cleavage and target site for the putative PmTCP2 mRNA was validated using RLM-5' RACE. The cleavage site was between the 10 thand 11 thbase of its interaction region. These results indicated that PmmiR319 a plays a role in flower bud development by regulating the target gene PmTCP2. The results of study provide a theoretical basis for the function of miR319 a to the development of Prumes mume
引文
[1]Zhang Y,Han J,Yu M,Ma R,Pervaiz T,Fang J.Characterization of target mRNAs for Prunus persica,microRNAs using an integrated strategy of PLM-RACE,PPM-RACE and qRT-PCR[J].Scientia Horticulturae,2014,170(3):8-16
[2]王海波,王莎莎,龚明.植物miRNA的分子特征及其在逆境中的响应机制[J].基因组学与应用生物学,2013,32(1):121-126
[3]Achard P,Herr A,Baulcombe D C,Harberd N P.Modulation of floral development by a gibberellin-regulated microRNA[J].Development,2004,131(14):3357-3365
[4]Aukerman M J,Sakai H.Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes[J].Plant Cell,2003,15(11):2730-2741
[5]Wang T,Pan H,Wang J,Yang W,Cheng T,Zhang Q.Identification and profiling of novel and conserved microRNAs during the flower opening process in Prunus mume via deep sequencing[J].Molecular Genetics and Genomics,2014,289(2):169-183
[6]Leung A K L,Sharp P A.MicroRNA functions in stress responses[J].Molecular Cell,2010,40(2):205-215
[7]Nag A,King S,Jack T.miR319a targeting of TCP4 is critical for petal growth and development in Arabidopsis[J].Proceedings of the National Academy of Sciences,2009,106(52):22534-22539
[8]陆奇丰,黄至欢.芸薹属物种(B.napus,B.rapa,B.oleracea)TCP基因家族全基因组鉴定和分析[J].分子植物育种,2016,14(12):3289-3298
[9]Danisman S.TCP transcription factors at the interface between environmental challenges and the plant’s growth responses[J].Frontiers in Plant Science,2016,7(406):1930
[10]Chuck G,Cigan A M,Saeteurn K,Hake S.The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA[J].Nature Genetics,2007,39(4):544-549
[11]褚孟嫄,房经贵.果梅文化[J].北京林业大学学报,2001,3(S1):47-49
[12]Jeong J T,Moon J H,Park K H,Shin C S.Isolation and characterization of a new compound from Prunus mume fruit that inhibits cancer cells[J].Journal of Agricultural&Food Chemistry,2006,54(6):2123-2128
[13]Gao Z,Shi T,Luo X Y,Zhang Z,Zhuang W,Wang L.Highthroughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot[J].BMC Genomics,2012,13(1):371
[14]蔡斌华,张计育,高志红,渠慎春,佟兆国,靡林.一种改良的提取草莓属叶片总RNA的方法[J].江苏农业学报,2008,24(6):875-877
[15]Wang X W,Xiong A S,Yao Q H,Zhen Z,Qiao Y S.Direct isolation of high-quality low molecular weight RNA of pear peel from the extraction mixture containing nucleic acid[J].Molecular Biotechnology,2010,44(1):61-65
[16]Tong Z G,Gao Z H,Wang F,Zhang Z.Selection of reliable reference genes for gene expression studies in peach using real-time PCR[J].BMC Molecular Biology,2009,10(1):1-13
[17]Ma C,Lu Y,Bai S,Zhang W,Duan X,Meng D.Cloning and characterization of miRNAs and their targets,including a novel miRNA-targeted NBS-LRR protein class gene in apple(Golden Delicious)[J].Molecular Plant,2014,7(1):218-230
[18]Meng Y J,Shao C G,Ma X X,Wang H.Introns targeted by plant microRNAs:a possible novel mechanism of gene regulation[J].Rice,2013,6(1):1-10
[19]Ercoli M F,Rojas A M,Debernardi J M,Palatnik J F,Rodriguez RE.Control of cell proliferation and elongation by miR396[J].Plant Signaling&Behavior,2016,11(6):e1184809
[20]Martin A,Adam H,Díazmendoza M,Zurczak M,Gonzálezschain ND,Suárezlópez P.Graft-transmissible induction of potato tuberization by the microRNA miR172[J].Development,2009,136(17):2873-2881
[21]Mao Y,He Y.MicroRNA319a-targeted Brassica rapa ssp.pekinensis TCP genes modulate head shape in chinese cabbage by differential cell division arrest in leaf regions[J].Plant Physiology,2014,164(2):710-720
[22]Rubiosomoza I,Weigel D.Coordination of flower maturation by a regulatory circuit of three microRNAs[J].PLo S Genetics,2013,9(3):e1003374
[23]Luo Y,Guo Z,Li L.Evolutionary conservation of microRNAregulatory programs in plant flower development[J].Developmental Biology,2013,380(2):133-144
[24]Amasino R,Mccourt P,Benning C.Seasonal and developmental timing of flowering[J].Plant Journal for Cell&Molecular Biology,2010,61(6):1001-1013
[25]Poethig R S.Small RNAs and developmental timing in plants[J].Current Opinion in Genetics&Development,2009,19(4):374-378
[26]Wang Z J,Huang J Q,Huang Y J,Li Z,Zheng B S.Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing[J].Planta,2012,236(2):613-621
[27]Thomson D W,Dinger M E.Endogenous microRNA sponges:evidence and controversy[J].Nature Reviews Genetics,2016,17(5):272-283
[28]Jones-Rhoades M W,Bartel D P.Computational identification of plant MicroRNAs and their targets,including a stress-induced miRNA[J].Molecular Cell,2004,14(6):787-799
[29]于丽丽,刘伟伟,方媛,周莹,王如意,李洋,周波.番茄LemiR390及其预测靶基因Le TAS3的鉴定与表达分析[J].园艺学报,2015,42(2):271-279
[2]王海波,王莎莎,龚明.植物miRNA的分子特征及其在逆境中的响应机制[J].基因组学与应用生物学,2013,32(1):121-126
[3]Achard P,Herr A,Baulcombe D C,Harberd N P.Modulation of floral development by a gibberellin-regulated microRNA[J].Development,2004,131(14):3357-3365
[4]Aukerman M J,Sakai H.Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes[J].Plant Cell,2003,15(11):2730-2741
[5]Wang T,Pan H,Wang J,Yang W,Cheng T,Zhang Q.Identification and profiling of novel and conserved microRNAs during the flower opening process in Prunus mume via deep sequencing[J].Molecular Genetics and Genomics,2014,289(2):169-183
[6]Leung A K L,Sharp P A.MicroRNA functions in stress responses[J].Molecular Cell,2010,40(2):205-215
[7]Nag A,King S,Jack T.miR319a targeting of TCP4 is critical for petal growth and development in Arabidopsis[J].Proceedings of the National Academy of Sciences,2009,106(52):22534-22539
[8]陆奇丰,黄至欢.芸薹属物种(B.napus,B.rapa,B.oleracea)TCP基因家族全基因组鉴定和分析[J].分子植物育种,2016,14(12):3289-3298
[9]Danisman S.TCP transcription factors at the interface between environmental challenges and the plant’s growth responses[J].Frontiers in Plant Science,2016,7(406):1930
[10]Chuck G,Cigan A M,Saeteurn K,Hake S.The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA[J].Nature Genetics,2007,39(4):544-549
[11]褚孟嫄,房经贵.果梅文化[J].北京林业大学学报,2001,3(S1):47-49
[12]Jeong J T,Moon J H,Park K H,Shin C S.Isolation and characterization of a new compound from Prunus mume fruit that inhibits cancer cells[J].Journal of Agricultural&Food Chemistry,2006,54(6):2123-2128
[13]Gao Z,Shi T,Luo X Y,Zhang Z,Zhuang W,Wang L.Highthroughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot[J].BMC Genomics,2012,13(1):371
[14]蔡斌华,张计育,高志红,渠慎春,佟兆国,靡林.一种改良的提取草莓属叶片总RNA的方法[J].江苏农业学报,2008,24(6):875-877
[15]Wang X W,Xiong A S,Yao Q H,Zhen Z,Qiao Y S.Direct isolation of high-quality low molecular weight RNA of pear peel from the extraction mixture containing nucleic acid[J].Molecular Biotechnology,2010,44(1):61-65
[16]Tong Z G,Gao Z H,Wang F,Zhang Z.Selection of reliable reference genes for gene expression studies in peach using real-time PCR[J].BMC Molecular Biology,2009,10(1):1-13
[17]Ma C,Lu Y,Bai S,Zhang W,Duan X,Meng D.Cloning and characterization of miRNAs and their targets,including a novel miRNA-targeted NBS-LRR protein class gene in apple(Golden Delicious)[J].Molecular Plant,2014,7(1):218-230
[18]Meng Y J,Shao C G,Ma X X,Wang H.Introns targeted by plant microRNAs:a possible novel mechanism of gene regulation[J].Rice,2013,6(1):1-10
[19]Ercoli M F,Rojas A M,Debernardi J M,Palatnik J F,Rodriguez RE.Control of cell proliferation and elongation by miR396[J].Plant Signaling&Behavior,2016,11(6):e1184809
[20]Martin A,Adam H,Díazmendoza M,Zurczak M,Gonzálezschain ND,Suárezlópez P.Graft-transmissible induction of potato tuberization by the microRNA miR172[J].Development,2009,136(17):2873-2881
[21]Mao Y,He Y.MicroRNA319a-targeted Brassica rapa ssp.pekinensis TCP genes modulate head shape in chinese cabbage by differential cell division arrest in leaf regions[J].Plant Physiology,2014,164(2):710-720
[22]Rubiosomoza I,Weigel D.Coordination of flower maturation by a regulatory circuit of three microRNAs[J].PLo S Genetics,2013,9(3):e1003374
[23]Luo Y,Guo Z,Li L.Evolutionary conservation of microRNAregulatory programs in plant flower development[J].Developmental Biology,2013,380(2):133-144
[24]Amasino R,Mccourt P,Benning C.Seasonal and developmental timing of flowering[J].Plant Journal for Cell&Molecular Biology,2010,61(6):1001-1013
[25]Poethig R S.Small RNAs and developmental timing in plants[J].Current Opinion in Genetics&Development,2009,19(4):374-378
[26]Wang Z J,Huang J Q,Huang Y J,Li Z,Zheng B S.Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing[J].Planta,2012,236(2):613-621
[27]Thomson D W,Dinger M E.Endogenous microRNA sponges:evidence and controversy[J].Nature Reviews Genetics,2016,17(5):272-283
[28]Jones-Rhoades M W,Bartel D P.Computational identification of plant MicroRNAs and their targets,including a stress-induced miRNA[J].Molecular Cell,2004,14(6):787-799
[29]于丽丽,刘伟伟,方媛,周莹,王如意,李洋,周波.番茄LemiR390及其预测靶基因Le TAS3的鉴定与表达分析[J].园艺学报,2015,42(2):271-279