PBU和6-BA对尾叶桉愈伤组织POD基因表达及酶活性的影响
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摘要
为了探讨脲类细胞分裂素PBU和嘌呤类细胞分裂素6-BA对尾叶桉愈伤分化、愈伤组织POD基因表达及酶活性的影响,将尾叶桉幼苗下胚轴切段接种在添加PBU+IAA、6-BA+IAA和PBU+6-BA+IAA三种激素组合的SPCa培养基中,通过统计愈伤组织分化情况、检测POD基因表达差异和酶活性变化来开展研究。6-BA+IAA的组合条件下,愈伤组织褐化率高达84.71%;PBU+IAA组合下,愈伤组织褐化率为30.56%;PBU+6-BA+IAA组合时,褐化率最低,为24.09%,胚性愈伤组织时的比例最高,达47.73%。与6-BA相比,PBU诱导出的愈伤POD酶活性显著升高。通过实时定量PCR (real-time quantitative PCR, qPCR)检测6个POD基因的表达变化,设6-BA+IAA诱导所得愈伤的相对表达量为1,PBU+IAA诱导所得愈伤中BP1,BP4,BP5表达上调,分别为1.88、1.63、2.01;PBU+6-BA+IAA诱导所得愈伤中BP3、BP4、BP6表达上调,分别为1.88、1.96、1.93。从实验结果分析,PBU和6-BA有协同效应,通过增强POD酶活性,减轻褐化、促进胚性愈伤分化。PBU和6-BA对不同POD同工酶表达的影响不同。本研究可为深入研究PBU和6-BA协同作用的分子机理提供参考。
In order to investigate the effects of urea-cytokinin PBU and purine cytokinin 6-BA on callus differentiation, the callus POD genes expression, and peroxidase activity, this study inoculated the hypocotyls fragments of Eucalyptus urophylla into SPCa medium supplemented with PBU +IAA, 6-BA +IAA or PBU +6-BA +IAA. We carried out the research by the statistics of the condition in callus differentiation, the detection of differentially expressed POD genes, and the enzymes activity. Induced by the combination of 6-BA and IAA, the browning rate of callus was up to 84.71%, and that of PBU+IAA was only 30.56%. Under the combination of PBU+6-BA+IAA,the browning rate was the lowest(24.09%) and the proportion of embryogenic callus was the highest(47.73%).Compared with 6-BA, the POD activity in the callus induced by PBU increased significantly. Then, the expression changes of six POD genes were detected by real-time quantitative PCR. With the transcriptional level of the same gene in the callus induced by 6-BA be normalized to one, the up regulated expression of BP1, BP4, BP5 were1.88, 1.63 and 2.01, respectively, in the callus induced by PBU. And the up regulated expression of BP3, BP4, BP6 were 1.88, 1.96 and 1.93, respectively, in the calli induced by the combination of PBU and 6-BA. The results suggested that PBU and 6-BA might have synergistic effect, which could reduce browning and promote embryogenic callus differentiation by enhancing the activity of POD. The effects of PBU and 6-BA on the expression of different POD isozymes were different. This reaesrch would provide a reference for the further study of the molecular mechanism of PBU and 6-BA synergism.
In order to investigate the effects of urea-cytokinin PBU and purine cytokinin 6-BA on callus differentiation, the callus POD genes expression, and peroxidase activity, this study inoculated the hypocotyls fragments of Eucalyptus urophylla into SPCa medium supplemented with PBU +IAA, 6-BA +IAA or PBU +6-BA +IAA. We carried out the research by the statistics of the condition in callus differentiation, the detection of differentially expressed POD genes, and the enzymes activity. Induced by the combination of 6-BA and IAA, the browning rate of callus was up to 84.71%, and that of PBU+IAA was only 30.56%. Under the combination of PBU+6-BA+IAA,the browning rate was the lowest(24.09%) and the proportion of embryogenic callus was the highest(47.73%).Compared with 6-BA, the POD activity in the callus induced by PBU increased significantly. Then, the expression changes of six POD genes were detected by real-time quantitative PCR. With the transcriptional level of the same gene in the callus induced by 6-BA be normalized to one, the up regulated expression of BP1, BP4, BP5 were1.88, 1.63 and 2.01, respectively, in the callus induced by PBU. And the up regulated expression of BP3, BP4, BP6 were 1.88, 1.96 and 1.93, respectively, in the calli induced by the combination of PBU and 6-BA. The results suggested that PBU and 6-BA might have synergistic effect, which could reduce browning and promote embryogenic callus differentiation by enhancing the activity of POD. The effects of PBU and 6-BA on the expression of different POD isozymes were different. This reaesrch would provide a reference for the further study of the molecular mechanism of PBU and 6-BA synergism.
引文
Bradford M.M.,1976,A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,Anal.Biochem.,71(1-2):248-254
Hothorn M.,Dabi T.,and Chory J.,2011,Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase4,Nat.Chem.Biol.,7:766-768
Huang Z.C.,Ouyang L.J.,Li Z.F.,and Zeng F.H.,2014,A urea-type cytokinin,2-Cl-PBU,stimulates adventitious bud formation of Eucalyptus urophylla by repressing transcription of rboh1 gene,Plant Cell,119(2):359-368
Huang Z.C.,Ouyang L.J.,Zeng F.H.,and Yu Q.M.,2010,Relationship between bud differentiation and several physiological and biochemical indexes of three types of Eucalyptus urophylla S.T.Blake callus,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),46(2):147-149(黄真池,欧阳乐军,曾富华,余秋梅,2010,尾叶桉的不同类型愈伤组织芽分化与某些相关生理生化指标的关系,植物生理学通讯,46(2):147-149)
Huang Z.C.,Zeng F.H.,and Lu X.Y.,2010,Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls,Biologia Plantarum,54(1):131-134
Meng M.,Huang X.F.,Li L.,Luo Y.C.,and Wang W.S.,2017,Effects of arsenic stress on activities of antioxidant enzymes of Eucalyptus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(12):5289-5295(蒙敏,黄雪芬,李磊,罗宇晨,王维生,2017,砷胁迫对桉树抗氧化酶活性的影响,基因组学与应用生物学,36(12):5289-5295)
Ouyang L.J.,and Zeng F.H.,2008,Advances in molecular breeding of Eucalyptus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(6):1146-1152(欧阳乐军,曾富华,2008,桉树分子育种研究进展,分子植物育种,6(6):1146-1152)
Pfaffl M.W.,Horgan G.W.,and Dempfle L.,2002,Relative expression software tool(REST)for group-wise comparison and statistical analysis of relative expression results in real-time PCR,Nucleic Acids Res.,30(9):e36
Ricci A.,and Bertoletti C.,2009,Urea derivatives on the move:cytokininlike activity and adventitious rooting enhancement depend on chemical structure,Plant Biol.,11:262-272
Wang J.,Tian C.H.,Zhang C.,Shi B.H.,Cao X.W.,Zhang T.Q.,Zhao Z.,Wang J.W.,and Jiao Y.L.,2017,Cytokinin signaling activates WUSCHEL expression during axillary meristem initiation,Plant Cell,29(6):1373-1387
Xu X.R.,Deng M.Q.,Lin Z.H.,Liao W.T.,Li L.M.,Zhou T.,and Ouyang L.J.,2017,Study on the relationship between the activity difference of resistance-related enzyme of different calluses of E.uroqhylla×E.grandls and the differentiation of adventitious bud,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):798-804(徐锡荣,邓毛琴,林壮辉,廖婉婷,李莉梅,周天,欧阳乐军,2017,尾巨桉不同类型愈伤组织抗性相关酶活性差异与不定芽分化关系研究,基因组学与应用生物学,36(2):798-804)
Zhang Z.J.,Tucker E.,Hermann M.,and Laux T.,2017,Amolecular framework for the embryonic initiation of shoot meristem stem cells,Developmental Cell,40(3):264-277
Hothorn M.,Dabi T.,and Chory J.,2011,Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase4,Nat.Chem.Biol.,7:766-768
Huang Z.C.,Ouyang L.J.,Li Z.F.,and Zeng F.H.,2014,A urea-type cytokinin,2-Cl-PBU,stimulates adventitious bud formation of Eucalyptus urophylla by repressing transcription of rboh1 gene,Plant Cell,119(2):359-368
Huang Z.C.,Ouyang L.J.,Zeng F.H.,and Yu Q.M.,2010,Relationship between bud differentiation and several physiological and biochemical indexes of three types of Eucalyptus urophylla S.T.Blake callus,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),46(2):147-149(黄真池,欧阳乐军,曾富华,余秋梅,2010,尾叶桉的不同类型愈伤组织芽分化与某些相关生理生化指标的关系,植物生理学通讯,46(2):147-149)
Huang Z.C.,Zeng F.H.,and Lu X.Y.,2010,Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls,Biologia Plantarum,54(1):131-134
Meng M.,Huang X.F.,Li L.,Luo Y.C.,and Wang W.S.,2017,Effects of arsenic stress on activities of antioxidant enzymes of Eucalyptus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(12):5289-5295(蒙敏,黄雪芬,李磊,罗宇晨,王维生,2017,砷胁迫对桉树抗氧化酶活性的影响,基因组学与应用生物学,36(12):5289-5295)
Ouyang L.J.,and Zeng F.H.,2008,Advances in molecular breeding of Eucalyptus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),6(6):1146-1152(欧阳乐军,曾富华,2008,桉树分子育种研究进展,分子植物育种,6(6):1146-1152)
Pfaffl M.W.,Horgan G.W.,and Dempfle L.,2002,Relative expression software tool(REST)for group-wise comparison and statistical analysis of relative expression results in real-time PCR,Nucleic Acids Res.,30(9):e36
Ricci A.,and Bertoletti C.,2009,Urea derivatives on the move:cytokininlike activity and adventitious rooting enhancement depend on chemical structure,Plant Biol.,11:262-272
Wang J.,Tian C.H.,Zhang C.,Shi B.H.,Cao X.W.,Zhang T.Q.,Zhao Z.,Wang J.W.,and Jiao Y.L.,2017,Cytokinin signaling activates WUSCHEL expression during axillary meristem initiation,Plant Cell,29(6):1373-1387
Xu X.R.,Deng M.Q.,Lin Z.H.,Liao W.T.,Li L.M.,Zhou T.,and Ouyang L.J.,2017,Study on the relationship between the activity difference of resistance-related enzyme of different calluses of E.uroqhylla×E.grandls and the differentiation of adventitious bud,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):798-804(徐锡荣,邓毛琴,林壮辉,廖婉婷,李莉梅,周天,欧阳乐军,2017,尾巨桉不同类型愈伤组织抗性相关酶活性差异与不定芽分化关系研究,基因组学与应用生物学,36(2):798-804)
Zhang Z.J.,Tucker E.,Hermann M.,and Laux T.,2017,Amolecular framework for the embryonic initiation of shoot meristem stem cells,Developmental Cell,40(3):264-277