矮牵牛海藻糖-6-磷酸合酶基因PhTPS6的克隆与表达分析
|
摘要
以矮牵牛(Petunia hybrida var.Mitchell)为材料,利用PCR方法克隆得到海藻糖-6-磷酸合酶基因6(TPS6)(Trehalose-6-phosphate synthase 6)的同源基因PhTPS6。生物信息学分析显示:该基因cDNA全长为2 571bp,编码856个氨基酸,推测PhTPS6蛋白的分子式为C4342H6838N1154O1276S48。利用实时荧光定量PCR分析PhTPS6在不同组织、不同处理下的表达特性。结果显示:PhTPS6基因在叶腋中表达量较高,而在叶片中的表达量最低;去顶6h引起PhTPS6的表达量升高至对照的14倍,24h后表达量显著下降。而去顶后施加生长素则抑制去顶对PhTPS6的调控。施加细胞分裂素6h后PhTPS6的表达水平显著上调至对照的16倍,随着处理时间的增加,其表达水平逐渐下降。低温处理12h以及盐胁迫处理12h导致PhTPS6表达量分别上调至对照的18.9倍和21.4倍,且随着时间的增加表达量持续上升。该研究表明PhTPS6在矮牵牛分枝发育及低温和盐胁迫中均具有重要作用。
The homologous gene PhTPS6 of TPS6(Trehalose-6-phosphate synthase 6)was cloned by PCR fromPetunia hybrida var.Mitchell.Bioinformatics analysis showed that the full-length cDNA of this gene was 2 571 bp,encoding 856 amino acids.It was speculated that the molecular formula of PhTPS6 protein was C4342 H6838 N1154 O1276 S48.Real-time quantitative PCR was used to analyze the expression characteristics of PhTPS6 under different tissues and treatments.The results showed that the expression level of PhTPS6 was higher in axillaries and lowest in leaves.The expression of PhTPS6 increased to 14 times of the control after 6 hof decapitation,and decreased significantly after24 h.The application of auxin to the top inhibited the regulation of PhTPS6 by decapitation.The expression level of PhTPS6 was significantly up-regulated to 16 times after application of cytokinin for6 h,and its expression gradually decreased with the increase of treatment time.After 12 hof low temperature treatment and 12 hof salt stress treatment,the expression level of PhTPS6 was up-regulated to 18.9 and 21.4 times of the control,respectively,and the expression level continued to increase with time.This study indicated that PhTPS6 played an important role in branch development,low temperature and salt stress of Petunia hybrida.
The homologous gene PhTPS6 of TPS6(Trehalose-6-phosphate synthase 6)was cloned by PCR fromPetunia hybrida var.Mitchell.Bioinformatics analysis showed that the full-length cDNA of this gene was 2 571 bp,encoding 856 amino acids.It was speculated that the molecular formula of PhTPS6 protein was C4342 H6838 N1154 O1276 S48.Real-time quantitative PCR was used to analyze the expression characteristics of PhTPS6 under different tissues and treatments.The results showed that the expression level of PhTPS6 was higher in axillaries and lowest in leaves.The expression of PhTPS6 increased to 14 times of the control after 6 hof decapitation,and decreased significantly after24 h.The application of auxin to the top inhibited the regulation of PhTPS6 by decapitation.The expression level of PhTPS6 was significantly up-regulated to 16 times after application of cytokinin for6 h,and its expression gradually decreased with the increase of treatment time.After 12 hof low temperature treatment and 12 hof salt stress treatment,the expression level of PhTPS6 was up-regulated to 18.9 and 21.4 times of the control,respectively,and the expression level continued to increase with time.This study indicated that PhTPS6 played an important role in branch development,low temperature and salt stress of Petunia hybrida.
引文
[1]ELBEIN A D,PAN Y T,PASTUSZAK I,et al.New insights on trehalose:a multifunctional molecule[J].Glycobiology,2003,13(4):17-27.
[2]IORDACHESCU M,IMAI R.Trehalose biosynthesis in response to abiotic stresses[J].Journal of Integrative Plant Biology,2008,50(10):1223-1229.
[3]GAZZARRINI S,TSAI A Y L.Trehalose-6-phosphate and SnRK1kinases in plant development and signaling:the emerging picture[J].Frontiers in Plant Science,2014,5:119.
[4]魏兵强,王兰兰,张茹,等.辣椒TPS家族成员的鉴定与CaTPS1的表达分析[J].园艺学报,2016,43(8):1504-1512.WEI B Q,WANG L L,ZHANG R,et al.Identification of TPS family members and expression analysis of CaTPS1in Capsicum[J].Acta Horticulyurae Sinica,2016,43(8):1504-1512.
[5]JIANG T,ZHAI H,WANG F B,et al.Cloning and characterization of a salt tolerance-associated gene encoding trehalose-6-phosphate synthase in sweetpotato[J].Journal of Integrative Agriculture,2014,13(8):1651-1661.
[6]GMEZ L D,GILDAY A,FEIL R,et al.AtTPS1-mediated trehalose 6-phosphate synthesis is essential for embryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells[J].The Plant Journal,2010,64(1):1-13.
[7]BARBIER F F,LUNN J E,BEVERIDGE C A.Ready,steady,go!A sugar hit starts the race to shoot branching[J].Current Opinion in Plant Biology,2015,25:39-45.
[8]XU Y,WANG Y,MATTSON N,et al.Genome-wide analysis of the Solanum tuberosum(potato)trehalose-6-phosphate synthase(TPS)gene family:evolution and differential expression during development and stress[J].BMC Genomics,2017,18(1):926.
[9]ZHOU B,FANG Y,FAN Y,et al.Expressional characterization of two class I trehalose-6-phosphate synthase genes in Hevea brasiliensis(para rubber tree)suggests a role in rubber production[J].New Forests,2017,48(4):513-526.
[10]O'HARA L E,PAUL M J,WINGLER A.How do sugars regulate plant growth and development?New insight into the role of trehalose-6-phosphate[J].Molecular Plant,2013,6(2):261-274.
[11]LEYMAN B,VAN DIJCK P,THEVELEIN J M.An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana[J].Trends in Plant Science,2001,6(11):510-513.
[12]BARRAZA A,CONTRERAS-CUBAS C,ESTRADA-NA-VARRETE G,et al.The classⅡtrehalose 6-phosphate synthase gene PvTPS9modulates trehalose metabolism in Phaseolus vulgaris nodules[J].Frontiers in Plant Science,2016,7:1589.
[13]RAMON M,DE SMET I V E,VANDESTEENE L,et al.Extensive expression regulation and lack of heterologous enzymatic activity of the classⅡtrehalose metabolism proteins fromArabidopsis thaliana[J].Plant,Cell&Environment,2009,32(8):1015-1132.
[14]CHARY S N,HICKS G R,CHOI Y G,et al.Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis[J].Plant Physiology,2008,146(1):97-107.
[15]BOMBARELY A,MOSER M,AMRAD A,et al.Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida[J].Nature Plants,2016,2(6):16074.
[16]董丽丽,龚凌燕,陈磊,等.石榴肉桂酰辅酶A还原酶基因的克隆与表达分析[J].南京农业大学学报,2016,39(5):747-753.DONG L L,GONG L Y,CHEN L,et al.Cloning and expression analysis of pomegranate cinnamoyl coenzyme Areductase gene[J].Journal of Nanjing Agricultural University,2016,39(5):747-753.
[17]GUO D,ZHANG J,WANG X,et al.The WRKY transcription factor WRKY71/EXB1controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis[J].The Plant Cell,2015,27(11):3112-3127.
[18]MLLER D,LEYSER O.Auxin,cytokinin and the control of shoot branching[J].Annals of Botany,2011,107(7):1203-1212.
[19]VANLAERE A.Trehalose,reserve and/or stress metabolite[J].FemS Microbiology Letters,1989,63(3):201-210.
[20]黄鹤,李卓夫,王晓楠,等.低温下冬小麦与中国春的TPS基因表达分析[J].核农学报,2016(7):1255-1262.HUANG H,LI ZH F,WANG X N,et al.Analysis of TPSgene expression in winter wheat and Chinese spring at low temperature[J].Journal of Nuclear Agricultural Sciences,2016(7):1255-1262.
[2]IORDACHESCU M,IMAI R.Trehalose biosynthesis in response to abiotic stresses[J].Journal of Integrative Plant Biology,2008,50(10):1223-1229.
[3]GAZZARRINI S,TSAI A Y L.Trehalose-6-phosphate and SnRK1kinases in plant development and signaling:the emerging picture[J].Frontiers in Plant Science,2014,5:119.
[4]魏兵强,王兰兰,张茹,等.辣椒TPS家族成员的鉴定与CaTPS1的表达分析[J].园艺学报,2016,43(8):1504-1512.WEI B Q,WANG L L,ZHANG R,et al.Identification of TPS family members and expression analysis of CaTPS1in Capsicum[J].Acta Horticulyurae Sinica,2016,43(8):1504-1512.
[5]JIANG T,ZHAI H,WANG F B,et al.Cloning and characterization of a salt tolerance-associated gene encoding trehalose-6-phosphate synthase in sweetpotato[J].Journal of Integrative Agriculture,2014,13(8):1651-1661.
[6]GMEZ L D,GILDAY A,FEIL R,et al.AtTPS1-mediated trehalose 6-phosphate synthesis is essential for embryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells[J].The Plant Journal,2010,64(1):1-13.
[7]BARBIER F F,LUNN J E,BEVERIDGE C A.Ready,steady,go!A sugar hit starts the race to shoot branching[J].Current Opinion in Plant Biology,2015,25:39-45.
[8]XU Y,WANG Y,MATTSON N,et al.Genome-wide analysis of the Solanum tuberosum(potato)trehalose-6-phosphate synthase(TPS)gene family:evolution and differential expression during development and stress[J].BMC Genomics,2017,18(1):926.
[9]ZHOU B,FANG Y,FAN Y,et al.Expressional characterization of two class I trehalose-6-phosphate synthase genes in Hevea brasiliensis(para rubber tree)suggests a role in rubber production[J].New Forests,2017,48(4):513-526.
[10]O'HARA L E,PAUL M J,WINGLER A.How do sugars regulate plant growth and development?New insight into the role of trehalose-6-phosphate[J].Molecular Plant,2013,6(2):261-274.
[11]LEYMAN B,VAN DIJCK P,THEVELEIN J M.An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana[J].Trends in Plant Science,2001,6(11):510-513.
[12]BARRAZA A,CONTRERAS-CUBAS C,ESTRADA-NA-VARRETE G,et al.The classⅡtrehalose 6-phosphate synthase gene PvTPS9modulates trehalose metabolism in Phaseolus vulgaris nodules[J].Frontiers in Plant Science,2016,7:1589.
[13]RAMON M,DE SMET I V E,VANDESTEENE L,et al.Extensive expression regulation and lack of heterologous enzymatic activity of the classⅡtrehalose metabolism proteins fromArabidopsis thaliana[J].Plant,Cell&Environment,2009,32(8):1015-1132.
[14]CHARY S N,HICKS G R,CHOI Y G,et al.Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis[J].Plant Physiology,2008,146(1):97-107.
[15]BOMBARELY A,MOSER M,AMRAD A,et al.Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida[J].Nature Plants,2016,2(6):16074.
[16]董丽丽,龚凌燕,陈磊,等.石榴肉桂酰辅酶A还原酶基因的克隆与表达分析[J].南京农业大学学报,2016,39(5):747-753.DONG L L,GONG L Y,CHEN L,et al.Cloning and expression analysis of pomegranate cinnamoyl coenzyme Areductase gene[J].Journal of Nanjing Agricultural University,2016,39(5):747-753.
[17]GUO D,ZHANG J,WANG X,et al.The WRKY transcription factor WRKY71/EXB1controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis[J].The Plant Cell,2015,27(11):3112-3127.
[18]MLLER D,LEYSER O.Auxin,cytokinin and the control of shoot branching[J].Annals of Botany,2011,107(7):1203-1212.
[19]VANLAERE A.Trehalose,reserve and/or stress metabolite[J].FemS Microbiology Letters,1989,63(3):201-210.
[20]黄鹤,李卓夫,王晓楠,等.低温下冬小麦与中国春的TPS基因表达分析[J].核农学报,2016(7):1255-1262.HUANG H,LI ZH F,WANG X N,et al.Analysis of TPSgene expression in winter wheat and Chinese spring at low temperature[J].Journal of Nuclear Agricultural Sciences,2016(7):1255-1262.