沙柳SpsLAS基因超表达对拟南芥营养生长的影响
|
摘要
用沙柳SpsLAS基因构建35S∷SpsLAS超表达载体并转化野生型拟南芥,对转基因拟南芥进行表型观察,利用荧光定量PCR,对分枝、生长素及细胞分裂素相关基因进行表达分析。结果显示:(1)成功构建35S∷SpsLAS超表达载体,并获得9株纯合转基因株系,且转基因株系的萌芽速率快于野生型(对照),生活周期也较长;其中7个株系表现为生长迅速、株高增加、莲座叶叶片增大、分枝增加,2个株系表现为矮化、分枝增加、育性降低等一系列变化。(2)荧光定量PCR显示,与对照相比24h时转基因株系幼苗生长素及细胞分裂素途径关键基因无明显变化,4d时各基因在各转基因株系呈上调趋势;30d时分枝相关基因RAX1、RAX3表达量均上调,而MAX1、MAX3、REV、AXR1无明显变化。研究表明,SpsLAS基因过表达对拟南芥株型、莲座叶有明显影响,该研究结果为进一步研究该基因对分枝调控机制奠定了基础。
LAS/LS subfamily plays an important role in the regulation of plant branching and development.The 35 s∷SpsLAS overexpression vector was constructed and transformed into wild type A.thaliana.These transgenic A.thaliana were screened by phenotype.The expressions of genes related to branching,auxin and cytokinin were analyzed by fluorescence quantitative PCR.The results showed that:(1)35 S∷SpsLAS overexpression vector was successfully transformed and nine homozygous transgenic lines of A.thaliana were obtained.The germination speed of transgenic lines were faster and life cycle was longer than that of wild type.Seven transgenic lines of them showed rapid growth,increased plant height,enlarged rosette leaves and more branches.Two lines showed a series of changes as dwarfing,more branches and lower fertility.(2)Fluorescent quantitative PCR showed that there were no significant changes for auxin and cytokinin pathway related genes at 24 hof seedling,while up-regulated at 4 d.Gene expressions of RAX1 and RAX3 related branching were up-regulated while MAX1,MAX3,REVand AXR1 had no obvious changes at 30 d.The research showed that the overexpression of SpsLAS gene has significanteffects on plant architecture and rosette leaves of A.thalianaand laid the foundation for further research on the regulation of branching mechanism.
LAS/LS subfamily plays an important role in the regulation of plant branching and development.The 35 s∷SpsLAS overexpression vector was constructed and transformed into wild type A.thaliana.These transgenic A.thaliana were screened by phenotype.The expressions of genes related to branching,auxin and cytokinin were analyzed by fluorescence quantitative PCR.The results showed that:(1)35 S∷SpsLAS overexpression vector was successfully transformed and nine homozygous transgenic lines of A.thaliana were obtained.The germination speed of transgenic lines were faster and life cycle was longer than that of wild type.Seven transgenic lines of them showed rapid growth,increased plant height,enlarged rosette leaves and more branches.Two lines showed a series of changes as dwarfing,more branches and lower fertility.(2)Fluorescent quantitative PCR showed that there were no significant changes for auxin and cytokinin pathway related genes at 24 hof seedling,while up-regulated at 4 d.Gene expressions of RAX1 and RAX3 related branching were up-regulated while MAX1,MAX3,REVand AXR1 had no obvious changes at 30 d.The research showed that the overexpression of SpsLAS gene has significanteffects on plant architecture and rosette leaves of A.thalianaand laid the foundation for further research on the regulation of branching mechanism.
引文
[1]STUURMAN J,JAGGI F,KUHLEMEIER C.Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells[J].Genes&Development,2002,16(17):2 213-2 218.
[2]MURAKAMI Y,MIWA H,IMAIZUMI-ANRAKU H,et al.Positional cloning identifies Lotus japonicus NSP2,aputative transcription factor of the GRAS family,required for NINand ENOD40gene expression in nodule initiation[J].DNA Research,2006,13(6):255-265.
[3]GRIFFITHS J,MURASE K,RIEU I,et al.Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis[J].Plant Cell,2006,18(12):3 399-3 414
[4]TONG H,JIN Y,LIU W,et al.Dwarf and low-tillering,a new member of the GRAS family,plays positive roles in brassinosteroid signaling in rice[J].Plant Journal for Cell&Molecular Biology,2009,58(5):803-816.
[5]MAYROSE M,EKENGREN S K,MELECH-BONFIL S,et al.A novel link between tomato GRAS genes,plant disease resistance and mechanical stress response[J].Molecular Plant Pathology,2006,7(6):593-604.
[6]GREB T,CLARENZ O,SCHAFER E,et al.Molecular analysis of the LATERAL SUPPRESSORgene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes&Development,2003,17(9):1 175-1 187.
[7]YUAN L H,PAN J S,WANG G,et al.The Cucumber Lateral Suppressor gene(CLS)is functionally associated with axillary meristem initiation[J].Plant Molecular Biology Reporter,2010,28(3):421-429.
[8]LI X,QIAN Q,FU Z,et al.Control of tillering in rice[J].Nature,2003,422(6 932):618-621.
[9]梁凌玲.青天葵分枝发育相关基因NfLs的克隆与表达分析[D].广州:广州中医药大学,2014.
[10]陈秀珍.两种芋兰分枝发育相关基因LS及其调控区的克隆与表达分析[D].广州:广州中医药大学,2016.
[11]路东晔,贺玉娇,金娜,等.沙柳SpsLAS基因克隆及生物信息学分析[J]分子植物育种,2017,15(2):483-491.LU D Y,HE Y J,JIN N,et al.Cloning and bioinformatics analysis of SpsLASgene in Salix psammophila[J].Molecular Plant Breeding,2017,15(2):483-491.
[12]王文娟,杨海峰,路东晔,等.拟南芥突变体pny的PCR鉴定及茎、下胚轴部位解剖结构变化分析[J].内蒙古农业大学学报(自然科学版),2016,37(1):40-46.WANG W J,YANG H F,LU D Y,et al.The PCR Identification and stem,hypocotyl structure changes of Arabidopsis mutant pny[J].Journal of Inner Mongolia Agricultural University,2016,37(1):40-46.
[13]陈芸,郑勇,刘霞,等.过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力[J].植物科学学报,2016,34(6):888-900.CHEN Y,ZHENG Y,LIU X,et al.Overexpression of the cotton CBF2 gene enhances salt and drought tolerance in Arabidopsis thaliana[J].Plant Science Journal,2016,34(6):888-900.
[14]YANG D H,YUN P Y,PARK S Y,et al.Cloning,characterization and expression of a Lateral suppressor-like gene from chrysanthemum(Dendranthema grandiflorum Kitamura)[J].Plant Physiology&Biochemistry,2005,43(12):1 044-1 051.
[15]YANG D H,SUN H J,GOH C H,et al.Cloning of a Zoysia ZjLsLand its overexpression to induce axillary meristem initiation and tiller formation in Arabidopsis and bentgrass[J].Plant Biology,2012,14(3):411-419.
[16]YANG M,YANG Q,FU T,et al.Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance[J].Plant Cell Reports,2011,30(3):373-388.
[17]MULLER D,SCHMITZ G,THERES K.Blindhomologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis[J].Plant Cell,2006,18(3):586-597.
[18]GREB T,CLARENZ O,SCHAFER E,et al.Molecular analysis of the LATERAL SUPPRESSORgene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes&Development,2003,17(9):1 175-1 187.
[19]RAMAN S,GREB T,PEAUCELLE A,et al.Interplay of miR164,CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSORcontrols axillary meristem formation in Arabidopsis thaliana[J].Plant Journal for Cell&Molecular Biology,2008,55(1):65-76.
[2]MURAKAMI Y,MIWA H,IMAIZUMI-ANRAKU H,et al.Positional cloning identifies Lotus japonicus NSP2,aputative transcription factor of the GRAS family,required for NINand ENOD40gene expression in nodule initiation[J].DNA Research,2006,13(6):255-265.
[3]GRIFFITHS J,MURASE K,RIEU I,et al.Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis[J].Plant Cell,2006,18(12):3 399-3 414
[4]TONG H,JIN Y,LIU W,et al.Dwarf and low-tillering,a new member of the GRAS family,plays positive roles in brassinosteroid signaling in rice[J].Plant Journal for Cell&Molecular Biology,2009,58(5):803-816.
[5]MAYROSE M,EKENGREN S K,MELECH-BONFIL S,et al.A novel link between tomato GRAS genes,plant disease resistance and mechanical stress response[J].Molecular Plant Pathology,2006,7(6):593-604.
[6]GREB T,CLARENZ O,SCHAFER E,et al.Molecular analysis of the LATERAL SUPPRESSORgene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes&Development,2003,17(9):1 175-1 187.
[7]YUAN L H,PAN J S,WANG G,et al.The Cucumber Lateral Suppressor gene(CLS)is functionally associated with axillary meristem initiation[J].Plant Molecular Biology Reporter,2010,28(3):421-429.
[8]LI X,QIAN Q,FU Z,et al.Control of tillering in rice[J].Nature,2003,422(6 932):618-621.
[9]梁凌玲.青天葵分枝发育相关基因NfLs的克隆与表达分析[D].广州:广州中医药大学,2014.
[10]陈秀珍.两种芋兰分枝发育相关基因LS及其调控区的克隆与表达分析[D].广州:广州中医药大学,2016.
[11]路东晔,贺玉娇,金娜,等.沙柳SpsLAS基因克隆及生物信息学分析[J]分子植物育种,2017,15(2):483-491.LU D Y,HE Y J,JIN N,et al.Cloning and bioinformatics analysis of SpsLASgene in Salix psammophila[J].Molecular Plant Breeding,2017,15(2):483-491.
[12]王文娟,杨海峰,路东晔,等.拟南芥突变体pny的PCR鉴定及茎、下胚轴部位解剖结构变化分析[J].内蒙古农业大学学报(自然科学版),2016,37(1):40-46.WANG W J,YANG H F,LU D Y,et al.The PCR Identification and stem,hypocotyl structure changes of Arabidopsis mutant pny[J].Journal of Inner Mongolia Agricultural University,2016,37(1):40-46.
[13]陈芸,郑勇,刘霞,等.过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力[J].植物科学学报,2016,34(6):888-900.CHEN Y,ZHENG Y,LIU X,et al.Overexpression of the cotton CBF2 gene enhances salt and drought tolerance in Arabidopsis thaliana[J].Plant Science Journal,2016,34(6):888-900.
[14]YANG D H,YUN P Y,PARK S Y,et al.Cloning,characterization and expression of a Lateral suppressor-like gene from chrysanthemum(Dendranthema grandiflorum Kitamura)[J].Plant Physiology&Biochemistry,2005,43(12):1 044-1 051.
[15]YANG D H,SUN H J,GOH C H,et al.Cloning of a Zoysia ZjLsLand its overexpression to induce axillary meristem initiation and tiller formation in Arabidopsis and bentgrass[J].Plant Biology,2012,14(3):411-419.
[16]YANG M,YANG Q,FU T,et al.Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance[J].Plant Cell Reports,2011,30(3):373-388.
[17]MULLER D,SCHMITZ G,THERES K.Blindhomologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis[J].Plant Cell,2006,18(3):586-597.
[18]GREB T,CLARENZ O,SCHAFER E,et al.Molecular analysis of the LATERAL SUPPRESSORgene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation[J].Genes&Development,2003,17(9):1 175-1 187.
[19]RAMAN S,GREB T,PEAUCELLE A,et al.Interplay of miR164,CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSORcontrols axillary meristem formation in Arabidopsis thaliana[J].Plant Journal for Cell&Molecular Biology,2008,55(1):65-76.