桑树中miR319的克隆及在雄花发育中表达分析
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摘要
为探究mi R319及靶基因在调控桑树雄花发育的分子机制,本研究通过石蜡切片技术判断桑树雄花芽发育时期及在线网站预测mi R319的靶基因。结果表明,桑树雄花芽发育分为未分化期(A1)、分化初期(A2)、花序分化期(A3)和总苞形成期(A4) 4个不同的阶段;桑树中mi R319家族存在mi R319a、mi R319b和mi R319c 3个成员,其中mi R319b与mi R319c位于同一条前体序列上,预测并获得了mi R319 6个靶基因。运用5'-RACE技术验证了mi R319a的4个靶基因,其切割位点主要集中在与mi RNA互补位点的第10和第11位碱基之间。采用RT-qPCR技术检测mi R319a及靶基因在雄花发育不同阶段的表达水平,发现mi R319a在雄花发育A2-A3阶段表达水平迅速降低,靶基因Morus012208、Morus008229、Morus008100与Morus005893在此阶段表达水平迅速升高,茉莉酸(JA)含量也迅速升高。同时,JA合成关键基因PLDα1和LOX5在雄花发育A2-A3阶段表达上调,表明mi R319通过调控靶基因影响JA合成来调控桑树花序分化。本研究为揭示桑树mi R319在JA途径中调控花发育中的分子调控机制奠定了一定的理论基础。
To explore the molecular mechanism of mi R319 and its target genes during male flower development in mulberry,histological technology was used to analyze the stage of flower bud development and the target genes of mi R319 were predicted through on-line software. We found that make flower development under went 4 different stages:undifferentiated period( A1),early differentiation stage( A2),inflorescence differentiation period( A3) and total pod formation period( A4). Three mi R319 members( mi R319 a、b、c) were found in mulberry genome,mi R319 b and mi R319 c were located on the same precursor sequence. A total of 6 target genes were found to be the targets of mi R319 in mulberry. Which were further validated via 5'-RACE,and the cleavage site was between the 10 thand 11 thbase. RTqPCR was used to detect the expression of mi R319 and it's targets during male flower development. The expression level of mi R319 reduced rapidly during A2-A3 stage,and target genes( Morus012208,Morus008229,Morus008100 and Morus005893) showed the opposite expression pattern. Meanwhile,the content of JA increased rapidly in this stage. JA synthetic component genes( PLDα1 and LOX5) also increased rapidly in the stage. It showed that mi R319 regulated the male flower development through influencing JA synthesis pathway by regulating the target gene. This study can help to reveal the molecular regulation mechanism of mi R319 regulating JA pathway during flower development.
To explore the molecular mechanism of mi R319 and its target genes during male flower development in mulberry,histological technology was used to analyze the stage of flower bud development and the target genes of mi R319 were predicted through on-line software. We found that make flower development under went 4 different stages:undifferentiated period( A1),early differentiation stage( A2),inflorescence differentiation period( A3) and total pod formation period( A4). Three mi R319 members( mi R319 a、b、c) were found in mulberry genome,mi R319 b and mi R319 c were located on the same precursor sequence. A total of 6 target genes were found to be the targets of mi R319 in mulberry. Which were further validated via 5'-RACE,and the cleavage site was between the 10 thand 11 thbase. RTqPCR was used to detect the expression of mi R319 and it's targets during male flower development. The expression level of mi R319 reduced rapidly during A2-A3 stage,and target genes( Morus012208,Morus008229,Morus008100 and Morus005893) showed the opposite expression pattern. Meanwhile,the content of JA increased rapidly in this stage. JA synthetic component genes( PLDα1 and LOX5) also increased rapidly in the stage. It showed that mi R319 regulated the male flower development through influencing JA synthesis pathway by regulating the target gene. This study can help to reveal the molecular regulation mechanism of mi R319 regulating JA pathway during flower development.
引文
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[2] Bai B,Bian H,Zeng Z,Hou N,Shi B,Wang J,Zhu M,Han N.mi R393-mediated auxin signaling regulation is involved in root elongation inhibition in response to toxic aluminum stress in barley[J]. Plant&Cell Physiology,2017,58(3):426-439
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[12] Hao J,Tu L,Hu H,Tan J,Deng F,Tang W,Nie Y,Zhang G X.GbTCP,a cotton TCP transcription factor,confers fibre elongation and root hair development by a complex regulating system[J].Journal of Experimental Botany,2012,63(17):6267-6281
[13] RubiosomozaⅠ,Weigel D. Coordination of flower maturation by a regulatory Circuit of three micro RNAs[J]. PLoS Genetics,2013,9(3):e1003374
[14]柯裕州,周金星,张旭东,孙启祥,左力.盐胁迫对桑树幼苗光合生理生态特性的影响[J].林业科学,2009,45(8):61-66
[15]罗子敬,孙宇涵,卢楠,李云.杨树耐盐机制及转基因研究进展[J].核农学报,2017,31(3):482-492
[16] He N,Zhang C,Qi X,Zhao S,Tao Y,Yang G,Lee T H,Wang X,Cai Q,Li D. Draft genome sequence of the mulberry tree Morus notabilis[J]. Nature Communications,2013,4(9):2445
[17] Llave C,Kasschau K D,Rector M A,Carrington J C. Endogenous and silencing-associated small RNAs in plants[J]. Plant Cell,2002,14(7):1605-1619
[18] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J]. Methods,2001,25(4):402-408
[19]孙志超,黄坚钦,王正加.与植物花发育相关的mi RNA研究进展[J].分子植物育种,2015,13(5):1177-1184
[20] Wang Z,Huang J,Sun Z,Zheng B. Identification of micro RNAs differentially expressed involved in male flower development[J].Functional&Integrative Genomics,2015,15(2):225-232
[21] Wang T, Pan H, Wang J, Yang W, Cheng T, Zhang Q.Identification and profiling of novel and conserved micro RNAs during the flower opening process in Prunus mume via deep sequencing[J].Molecular Genetics&Genomics,2014,289(2):169-183
[22] Dotto M,Gmez M S,Soto M S,Casati P. UV-B radiation delays flowering time through changes in the PRC2 complex activity and mi R156 levels in Arabidopsis thaliana[J]. Plant Cell&Environment,2018,41(6):1394-1406
[23] Debernardi J M,Lin H,Chuck G,Faris J D,Dubcovsky J.Micro RNA172 plays a crucial role in wheat spike morphogenesis and grain threshability[J]. Development,2017,144(11):1966-1975
[24] Yang F X,Zhu G F,Wang Z,Liu H L,Huang D. A putative mi R172-targeted CeAPETALA2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium[J].Genetics&Molecular Research Gmr,2015,14(4):12049-12061
[25] Fang Y,Xie K,Xiong L. Conserved mi R164-targeted NAC genes negatively regulate drought resistance in rice[J]. Journal of Experimental Botany,2014,65(8):2119-2135
[26] Mallory A C,Dugas DⅤ,Bartel D P,Bartel B. Micro RNA regulation of NAC-Domain targets is required for proper formation and separation of adjacent embryonic,vegetative,and floral organs[J]. Current Biology,2004,14(12):1035-1046
[27]孙志超,谢岩,杨帆,黄露,夏爱华,杨贵明,李季生.桑树mi R164及靶基因NAC在甘露醇和Na Cl胁迫中的表达分析[J].核农学报,2018,32(5):856-863
[28] Schommer C,Palatnik J F,Aggarwal P,Chtelat A,Cubas P,Farmer E E,Nath U,Weigel D. Control of Jasmonate biosynthesis and senescence by mi R319 targets[J]. PLoS Biology,2008,6(9):e230
[29] Chun H,Da Y,Dong H M,Xue L,Cheng J,Xiao B,Xian F,Zhu K,Cai Y, Li H. Overexpression of micro RNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice(Oryza sativa L.)[J]. Plant Cell&Environment,2013,36(12):2207-2218
[30] Han Y L,Seo J S,Cho J H,Jung H,Kim J K,Lee J S,Rhee S,Yang D C. Oryza sativa COI homologues restore Jasmonate signal transduction in Arabidopsis coi1-1 mutants[J]. PLoS One,2013,8(1):e52802
[2] Bai B,Bian H,Zeng Z,Hou N,Shi B,Wang J,Zhu M,Han N.mi R393-mediated auxin signaling regulation is involved in root elongation inhibition in response to toxic aluminum stress in barley[J]. Plant&Cell Physiology,2017,58(3):426-439
[3] Lee M H,Jeon H S,Kim H G,Park O K. An Arabidopsis NAC transcription factor NAC4 promotes pathogen‐induced cell death under negative regulation by micro RNA164[J]. New Phytologist,2017,214(1):343-360
[4] Huang W,Peng S,Xian Z,Lin D,Hu G,Yang L,Ren M,Li Z.Overexpression of a tomato mi R171 target gene SlGRAS24 impacts multiple agronomical traits via regulating gibberellin and auxin homeostasis[J]. Plant Biotechnology Journal,2016,15(4):472-488
[5] Sun Z,Zhang L,Wang Z. Genome-wide analysis of mi RNAs in Carya cathayensis[J]. BMC Plant Biology,2017,17(1):228
[6]舒李露,孙志超,张玮,付顺华,王正加.山核桃mi R169克隆及功能分析[J].核农学报,2017,31(8):1453-1462
[7]罗茂,张志明,高健,曾兴,潘光堂. mi R319在植物器官发育中的调控作用[J].遗传. 2011,33(11):1203-1211
[8] Nag A,King S,Jack T. mi R319a targeting of TCP4 is critical for petal growth and development in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America,2009,106(52):22534-22539
[9] Palatnik J F,Wollmann H,Schommer C,Schwab R,Boisbouvier J,Rodriguez R,Warthmann N,Allen E,Dezulian T,Huson D.Sequence and expression differences underlie functional specialization of Arabidopsis micro RNAs mi R159 and mi R319[J].Developmental Cell,2007,13(1):115-125
[10] Nicolas M,Cubas P. TCP factors:New kids on the signaling block[J]. Current Opinion in Plant Biology,2016,33:33-41
[11]闫志强,徐海,马作斌,高东昌,徐正进.籼稻与粳稻花时对茉莉酸甲酯(Me JA)响应的敏感性差异[J].中国农业科学,2014,47(13):2529-2540
[12] Hao J,Tu L,Hu H,Tan J,Deng F,Tang W,Nie Y,Zhang G X.GbTCP,a cotton TCP transcription factor,confers fibre elongation and root hair development by a complex regulating system[J].Journal of Experimental Botany,2012,63(17):6267-6281
[13] RubiosomozaⅠ,Weigel D. Coordination of flower maturation by a regulatory Circuit of three micro RNAs[J]. PLoS Genetics,2013,9(3):e1003374
[14]柯裕州,周金星,张旭东,孙启祥,左力.盐胁迫对桑树幼苗光合生理生态特性的影响[J].林业科学,2009,45(8):61-66
[15]罗子敬,孙宇涵,卢楠,李云.杨树耐盐机制及转基因研究进展[J].核农学报,2017,31(3):482-492
[16] He N,Zhang C,Qi X,Zhao S,Tao Y,Yang G,Lee T H,Wang X,Cai Q,Li D. Draft genome sequence of the mulberry tree Morus notabilis[J]. Nature Communications,2013,4(9):2445
[17] Llave C,Kasschau K D,Rector M A,Carrington J C. Endogenous and silencing-associated small RNAs in plants[J]. Plant Cell,2002,14(7):1605-1619
[18] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J]. Methods,2001,25(4):402-408
[19]孙志超,黄坚钦,王正加.与植物花发育相关的mi RNA研究进展[J].分子植物育种,2015,13(5):1177-1184
[20] Wang Z,Huang J,Sun Z,Zheng B. Identification of micro RNAs differentially expressed involved in male flower development[J].Functional&Integrative Genomics,2015,15(2):225-232
[21] Wang T, Pan H, Wang J, Yang W, Cheng T, Zhang Q.Identification and profiling of novel and conserved micro RNAs during the flower opening process in Prunus mume via deep sequencing[J].Molecular Genetics&Genomics,2014,289(2):169-183
[22] Dotto M,Gmez M S,Soto M S,Casati P. UV-B radiation delays flowering time through changes in the PRC2 complex activity and mi R156 levels in Arabidopsis thaliana[J]. Plant Cell&Environment,2018,41(6):1394-1406
[23] Debernardi J M,Lin H,Chuck G,Faris J D,Dubcovsky J.Micro RNA172 plays a crucial role in wheat spike morphogenesis and grain threshability[J]. Development,2017,144(11):1966-1975
[24] Yang F X,Zhu G F,Wang Z,Liu H L,Huang D. A putative mi R172-targeted CeAPETALA2-like gene is involved in floral patterning regulation of the orchid Cymbidium ensifolium[J].Genetics&Molecular Research Gmr,2015,14(4):12049-12061
[25] Fang Y,Xie K,Xiong L. Conserved mi R164-targeted NAC genes negatively regulate drought resistance in rice[J]. Journal of Experimental Botany,2014,65(8):2119-2135
[26] Mallory A C,Dugas DⅤ,Bartel D P,Bartel B. Micro RNA regulation of NAC-Domain targets is required for proper formation and separation of adjacent embryonic,vegetative,and floral organs[J]. Current Biology,2004,14(12):1035-1046
[27]孙志超,谢岩,杨帆,黄露,夏爱华,杨贵明,李季生.桑树mi R164及靶基因NAC在甘露醇和Na Cl胁迫中的表达分析[J].核农学报,2018,32(5):856-863
[28] Schommer C,Palatnik J F,Aggarwal P,Chtelat A,Cubas P,Farmer E E,Nath U,Weigel D. Control of Jasmonate biosynthesis and senescence by mi R319 targets[J]. PLoS Biology,2008,6(9):e230
[29] Chun H,Da Y,Dong H M,Xue L,Cheng J,Xiao B,Xian F,Zhu K,Cai Y, Li H. Overexpression of micro RNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice(Oryza sativa L.)[J]. Plant Cell&Environment,2013,36(12):2207-2218
[30] Han Y L,Seo J S,Cho J H,Jung H,Kim J K,Lee J S,Rhee S,Yang D C. Oryza sativa COI homologues restore Jasmonate signal transduction in Arabidopsis coi1-1 mutants[J]. PLoS One,2013,8(1):e52802