马铃薯StmiR166b靶基因的鉴定及其表达分析
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摘要
为阐明马铃薯(Solanum tuberosum L.)StmiR166b的生物学功能,以栽培品种‘甘农薯2号’试管苗为试验材料,采用定量PCR技术检测StmiR166b及其靶基因StHB14、StHB15在不同组织中的差异表达;利用pRS300质粒为模板,通过over-lapping PCR技术克隆了包含StmiR166b-StmiR166b*成熟体序列的前体结构,构建了其人工表达载体,通过根癌农杆菌(Agrobacterium tumefaciens)介导法转化马铃薯获得了转基因植株。结果表明,StmiR166b及其靶基因StHB14和StHB15在马铃薯根、茎和叶中均有表达,在转基因株系L1、L3中靶基因不同程度地下调表达;与非转基因对照相比,转基因株系的株高和根长变短,生长受到抑制。
In order to elucidate the biological function of StmiR166 b of potato(Solanum tuberosum L.),plantlets in vitro of potato cultivar‘Gannongshu 2'were used as dornors,the differential expression of StmiR166 b and its target genes StHB14 and StHB15 were determined in different tissues by real-time quantitative PCR. Furthmore,the pre-structure of StmiR166 b-StmiR166 b* muture sequence was cloned using pRS300 plasmid as a template and over-lapping PCR technology. The artificial expression vector was constructed and further the transgenic potato plants were obtained via Agrobacterium tumefaciensmediated method. The results showed that StmiR166 b and its target genes StHB14 and StHB15 were expressed in roots,stems and leaves,which were down-regulated expression in the transgenic lines L1 and L3. The growth of the transgenic lines was inhibited,and their plant height and root length were shorter cmopared with the untransgenic plants under normal growth conditions.
In order to elucidate the biological function of StmiR166 b of potato(Solanum tuberosum L.),plantlets in vitro of potato cultivar‘Gannongshu 2'were used as dornors,the differential expression of StmiR166 b and its target genes StHB14 and StHB15 were determined in different tissues by real-time quantitative PCR. Furthmore,the pre-structure of StmiR166 b-StmiR166 b* muture sequence was cloned using pRS300 plasmid as a template and over-lapping PCR technology. The artificial expression vector was constructed and further the transgenic potato plants were obtained via Agrobacterium tumefaciensmediated method. The results showed that StmiR166 b and its target genes StHB14 and StHB15 were expressed in roots,stems and leaves,which were down-regulated expression in the transgenic lines L1 and L3. The growth of the transgenic lines was inhibited,and their plant height and root length were shorter cmopared with the untransgenic plants under normal growth conditions.
引文
Axtell M J,Bartel D P.2005.Antiquity of microRNAs and their targets in land plants.The Plant Cell,17(6):1658-1673.
Bartel D P.2004.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,116(2):281-297.
Brandt R,Salla-Martret M,Bou-Torrent J,Musielak T,Stahl M,Lanz C,Ott F,Schmid M,Greb T,Schwarz M,Choi S B,Barton M K,Reinhart B J,Liu T,Quint M,Palauqui J C,Martinez-Garcia J F,Wenkel S.2012.Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses.The Plant Journal,72(1):31-42.
Chen X M.2005.microRNA biogenesis and function in plants.FEBS Letters,579:5923-5931.
Donaire L,Pedrola L,Rosa Rde L,Llave C.2011.High-throughput sequencing of RNA silencing-associated small RNAs in olive(Olea europaea L.).PLoS ONE,6(11):e27916.
Jones-Rhoades M W,Bartel D P,Bartel B.2006.MicroRNAs and their regulatory roles in plants.Annual Review of Plant Biology,57:19-53.
Khraiwesh B,Zhu J K,Zhu J.2012.Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants.Biochim Biophys Acta,1819(2):137-148.
Lee R C,Feinbaum R L,Ambros V 1993.The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell,75(5):843-854.
Li J,Guo G,Guo W,Guo G,Tong D,Ni Z,Sun Q,Yao Y.2012.miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize(Zea mays L.).BMC Plant Biology,12:220.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods,25(4):402-408.
Llave C,Kasschau K D,Rector M A,Carrington J C.2002.Endogenous and silencing-associated small RNAs in plants.The Plant Cell,14(7):1605-1619.
Ma Xinrui,Li Liang,Liu Jinhang,Yang Mengjie,Chen Jie,Liang Qin,Wu Shaohua,Li Yongyu.2018.Identification and differentially expressed analysis of microRNA associated with dormancy of pear flower buds.Acta Horticulturae Sinica,45(11):2089-2105.(in Chinese)马鑫瑞,李亮,刘瑾航,杨梦洁,陈洁,梁沁,吴少华,李永裕.2018.梨花芽休眠相关miRNA的鉴定和差异表达分析.园艺学报,45(11):2089-2105.
Magnani E,Barton M K.201 1.A per-ARNT-sim-like sensor domain uniquely regulates the activity of the homeodomain leucine zipper transcription factor REVOLUTA in Arabidopsis.The Plant Cell,23(2):567-582.
Ohashi-Ito K,Fukuda H.2003.HD-zipⅢhomeobox genes that include a novel member,ZeHB-13(Zinnia)/ATHB-15(Arabidopsis),are involved in procambium and xylem cell differentiation.Plant&Cell Physiology,44(12):1350-1358.
Ossowski S,Schwab R,Weigel D.2008.Gene silencing in plants using artificial microRNAs and other small RNAs.The Plant Journal,53(4):674-690.
Prigge M J,Otsuga D,Alonso J M,Ecker J R,Drews G N,Clark S E.2005.ClassⅢhomeodomain-leucine zipper gene family members have overlapping,antagonistic,and distinct roles in Arabidopsis development.The Plant Cell,17(1):61-76.
Qiu C X,Xie F L,Zhu Y Y,Guo K,Huang S Q,Nie L,Yang Z M.2007.Computational identification of microRNAs and their targets in Gossypium hirsutum expressed sequence tags.Gene,395(1-2):49-61.
Reinhart B J,Weinstein E G,Rhoades M W,Bartel B,Bartel D P.2002.MicroRNAs in plants.Genes&Development,16(13):1616-1626.
Schwab R,Ossowski S,Riester M,Warthmann N,Weigel D.2006.Highly specific gene silencing by artificial microRNAs in Arabidopsis.The Plant Cell,18(5):1121-1133.
Sessa G,Steindler C,Morelli G,Ruberti I.1998.The Arabidopsis Athb-8,-9 and-14 genes are members of a small gene family coding for highly related HD-ZIP proteins.Plant Molecular Biology,38(4):609-622.
Singh A,Singh S,Panigrahi K C,Reski R,Sarkar A K.2014.Balanced activity of microRNA166/165 and its target transcripts from the classⅢhomeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana.Plant Cell Reports,33(6):945-953.
Wang Xiao,Liu Yang,Chen Hai-yin,Liu Kun,Qi Ming-fang,Li Tian-lai,Liu Yu-feng.2016.The effect of low night temperature on miRNA expression in mutant and wild type tomato leaf.Acta Horticulturae Sinica,43(12):2369-2379.(in Chinese)王孝,刘阳,陈海银,刘琨,齐明芳,李天来,刘玉凤.2016.低夜温下番茄ABA缺失突变体和野生型叶片中miRNA差异表达分析.园艺学报,43(12):2369-2379.
Wang Xingru,Li Wenjing,Chen Bingxing,Liu Tao,Shang Wei,Lai Zhongxiong,Guo Rongfang.2019.Analysis of miRNA in water spinach(Ipomoea aquatica)under long-time high temperature.Acta Horticulturae Sinica,46(3):486-498.(in Chinese)王杏茹,李文静,陈冰星,刘涛,尚维,赖钟雄,郭容芳.2019.蕹菜耐受长时间高温后的imiRNA分析.园艺学报,46(3):486-498.
Williams L,Grigg S P,Xie M,Christensen S,Fletcher J C.2005.Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes.Development,132(16):3657-3668.
Zhang B,Pan X,Cannon C H,Cobb G P,Anderson T A.2006.Conservation and divergence of plant microRNA genes.The Plant Journal,46(2):243-259.
Zhang X,Zou Z,Gong P,Zhang J,Ziaf K,Li H,Xiao F,Ye Z.2011.Over-expression of microRNA 169 confers enhanced drought tolerance to tomato.Biotechnology Letters,33(2):403-409.
Zhou G K,Kubo M,Zhong R,Demura T,Ye Z H.2007.Overexpression of miR165 affects apical meristem formation,organ polarity establishment and vascular development in Arabidopsis.Plant&Cell Physiology,48(3):391-404.
Zhang Ning,Si Huaijun,Wang Di.2005.Cloning of rd29A gene promoter from Arabidopsis thaliana and its application in stress-resistance transgenic potato.Acta Agronomica Sinica,31(2):159-164.(in Chinese)张宁,司怀军,王蒂.2005.拟南芥rd291基因启动子克隆及其在马铃薯抗胁迫转基因中的应用.作物学报,31(2):159-164.
Zhang Qinglin,Su Liyao,Li Xue,Zhang Shuting,Xu Xiaoping,Chen Xiaohui,Wang Peiyu,Li Rong,Zhang Zihao,Chen Yukun,Lai Zhongxiong,Lin Yuling.2018.Cloning and expression analysis of miR166 primary gene during the early stage of somatic embryogenesis of longan.Acta Horticulturae Sinica,45(8):1501-1512.(in Chinese)张清林,苏立遥,厉雪,张舒婷,徐小萍,陈晓慧,王培育,李蓉,张梓浩,陈裕坤,赖钟雄,林玉玲.2018.龙眼体胚发生早期miR166初级体的克隆与表达分析.园艺学报,45(8):1501-1512.
Bartel D P.2004.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell,116(2):281-297.
Brandt R,Salla-Martret M,Bou-Torrent J,Musielak T,Stahl M,Lanz C,Ott F,Schmid M,Greb T,Schwarz M,Choi S B,Barton M K,Reinhart B J,Liu T,Quint M,Palauqui J C,Martinez-Garcia J F,Wenkel S.2012.Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses.The Plant Journal,72(1):31-42.
Chen X M.2005.microRNA biogenesis and function in plants.FEBS Letters,579:5923-5931.
Donaire L,Pedrola L,Rosa Rde L,Llave C.2011.High-throughput sequencing of RNA silencing-associated small RNAs in olive(Olea europaea L.).PLoS ONE,6(11):e27916.
Jones-Rhoades M W,Bartel D P,Bartel B.2006.MicroRNAs and their regulatory roles in plants.Annual Review of Plant Biology,57:19-53.
Khraiwesh B,Zhu J K,Zhu J.2012.Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants.Biochim Biophys Acta,1819(2):137-148.
Lee R C,Feinbaum R L,Ambros V 1993.The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell,75(5):843-854.
Li J,Guo G,Guo W,Guo G,Tong D,Ni Z,Sun Q,Yao Y.2012.miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize(Zea mays L.).BMC Plant Biology,12:220.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods,25(4):402-408.
Llave C,Kasschau K D,Rector M A,Carrington J C.2002.Endogenous and silencing-associated small RNAs in plants.The Plant Cell,14(7):1605-1619.
Ma Xinrui,Li Liang,Liu Jinhang,Yang Mengjie,Chen Jie,Liang Qin,Wu Shaohua,Li Yongyu.2018.Identification and differentially expressed analysis of microRNA associated with dormancy of pear flower buds.Acta Horticulturae Sinica,45(11):2089-2105.(in Chinese)马鑫瑞,李亮,刘瑾航,杨梦洁,陈洁,梁沁,吴少华,李永裕.2018.梨花芽休眠相关miRNA的鉴定和差异表达分析.园艺学报,45(11):2089-2105.
Magnani E,Barton M K.201 1.A per-ARNT-sim-like sensor domain uniquely regulates the activity of the homeodomain leucine zipper transcription factor REVOLUTA in Arabidopsis.The Plant Cell,23(2):567-582.
Ohashi-Ito K,Fukuda H.2003.HD-zipⅢhomeobox genes that include a novel member,ZeHB-13(Zinnia)/ATHB-15(Arabidopsis),are involved in procambium and xylem cell differentiation.Plant&Cell Physiology,44(12):1350-1358.
Ossowski S,Schwab R,Weigel D.2008.Gene silencing in plants using artificial microRNAs and other small RNAs.The Plant Journal,53(4):674-690.
Prigge M J,Otsuga D,Alonso J M,Ecker J R,Drews G N,Clark S E.2005.ClassⅢhomeodomain-leucine zipper gene family members have overlapping,antagonistic,and distinct roles in Arabidopsis development.The Plant Cell,17(1):61-76.
Qiu C X,Xie F L,Zhu Y Y,Guo K,Huang S Q,Nie L,Yang Z M.2007.Computational identification of microRNAs and their targets in Gossypium hirsutum expressed sequence tags.Gene,395(1-2):49-61.
Reinhart B J,Weinstein E G,Rhoades M W,Bartel B,Bartel D P.2002.MicroRNAs in plants.Genes&Development,16(13):1616-1626.
Schwab R,Ossowski S,Riester M,Warthmann N,Weigel D.2006.Highly specific gene silencing by artificial microRNAs in Arabidopsis.The Plant Cell,18(5):1121-1133.
Sessa G,Steindler C,Morelli G,Ruberti I.1998.The Arabidopsis Athb-8,-9 and-14 genes are members of a small gene family coding for highly related HD-ZIP proteins.Plant Molecular Biology,38(4):609-622.
Singh A,Singh S,Panigrahi K C,Reski R,Sarkar A K.2014.Balanced activity of microRNA166/165 and its target transcripts from the classⅢhomeodomain-leucine zipper family regulates root growth in Arabidopsis thaliana.Plant Cell Reports,33(6):945-953.
Wang Xiao,Liu Yang,Chen Hai-yin,Liu Kun,Qi Ming-fang,Li Tian-lai,Liu Yu-feng.2016.The effect of low night temperature on miRNA expression in mutant and wild type tomato leaf.Acta Horticulturae Sinica,43(12):2369-2379.(in Chinese)王孝,刘阳,陈海银,刘琨,齐明芳,李天来,刘玉凤.2016.低夜温下番茄ABA缺失突变体和野生型叶片中miRNA差异表达分析.园艺学报,43(12):2369-2379.
Wang Xingru,Li Wenjing,Chen Bingxing,Liu Tao,Shang Wei,Lai Zhongxiong,Guo Rongfang.2019.Analysis of miRNA in water spinach(Ipomoea aquatica)under long-time high temperature.Acta Horticulturae Sinica,46(3):486-498.(in Chinese)王杏茹,李文静,陈冰星,刘涛,尚维,赖钟雄,郭容芳.2019.蕹菜耐受长时间高温后的imiRNA分析.园艺学报,46(3):486-498.
Williams L,Grigg S P,Xie M,Christensen S,Fletcher J C.2005.Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes.Development,132(16):3657-3668.
Zhang B,Pan X,Cannon C H,Cobb G P,Anderson T A.2006.Conservation and divergence of plant microRNA genes.The Plant Journal,46(2):243-259.
Zhang X,Zou Z,Gong P,Zhang J,Ziaf K,Li H,Xiao F,Ye Z.2011.Over-expression of microRNA 169 confers enhanced drought tolerance to tomato.Biotechnology Letters,33(2):403-409.
Zhou G K,Kubo M,Zhong R,Demura T,Ye Z H.2007.Overexpression of miR165 affects apical meristem formation,organ polarity establishment and vascular development in Arabidopsis.Plant&Cell Physiology,48(3):391-404.
Zhang Ning,Si Huaijun,Wang Di.2005.Cloning of rd29A gene promoter from Arabidopsis thaliana and its application in stress-resistance transgenic potato.Acta Agronomica Sinica,31(2):159-164.(in Chinese)张宁,司怀军,王蒂.2005.拟南芥rd291基因启动子克隆及其在马铃薯抗胁迫转基因中的应用.作物学报,31(2):159-164.
Zhang Qinglin,Su Liyao,Li Xue,Zhang Shuting,Xu Xiaoping,Chen Xiaohui,Wang Peiyu,Li Rong,Zhang Zihao,Chen Yukun,Lai Zhongxiong,Lin Yuling.2018.Cloning and expression analysis of miR166 primary gene during the early stage of somatic embryogenesis of longan.Acta Horticulturae Sinica,45(8):1501-1512.(in Chinese)张清林,苏立遥,厉雪,张舒婷,徐小萍,陈晓慧,王培育,李蓉,张梓浩,陈裕坤,赖钟雄,林玉玲.2018.龙眼体胚发生早期miR166初级体的克隆与表达分析.园艺学报,45(8):1501-1512.