墨兰‘达摩’原叶绿素酸酯氧化还原酶基因CsPORB功能的初步研究
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摘要
为了解墨兰‘达摩’(Cymbidium sinense ‘Dharma’)的POR基因功能,从其嫩叶中克隆得到CsPORB基因。CsPORB的开放阅读框长度为1 200 bp,编码399个氨基酸,CsPORB分子量为43.13 kDa,理论等电点9.30;Cs PORB与其他物种的POR蛋白序列高度同源,在进化关系上较为保守;qRT-PCR表达分析表明,CsPORB在墨兰不同发育阶段和不同组织中的表达有明显差异,其集中于生长旺盛的嫩叶中表达。经高表达转基因表型验证,35S:CsPORB转拟南芥(Arabidopsis thaliana)植株中PORB及其下游叶绿素合成代谢酶活性显著提高,叶绿素含量增加,尤其在黑暗处理1周后,35S:CsPORB转基因植株仍表现持绿表型。因此,CsPORB基因可能在墨兰‘达摩’叶绿素合成代谢中发挥重要作用。
In order to understand the function of POR gene in Cymbidium sinense 'Dharma', one POR gene family member CsPORB was cloned from young leaves. The CsPORB gene contained an open reading frame of1 200 bp, which encoded a protein of 399 amino acids with molecular weight of 43.13 kDa and theoretical isoelectric point of 9.30. Sequence alignment showed that CsPORB was highly homologous to the sequence of POR family protein of other species, indicating conservative evolution of this gene family. The CsPORB expression showed obviously difference in different developmental stages and tissues, and high expression in growing young leaves. Furthermore, both of the PORB and its downstream chlorophyll biosynthesis enzyme activity increased in transgenic 35 S:CsPORB Arabidopsis, as well as chlorophyll content, especially 35 S:CsPORB transgenic plants remained green under dark for one week. Therefore, CsPORB could play an important roles in chlorophyll synthesis pathway of Cymbidium sinense 'Dharma'.
In order to understand the function of POR gene in Cymbidium sinense 'Dharma', one POR gene family member CsPORB was cloned from young leaves. The CsPORB gene contained an open reading frame of1 200 bp, which encoded a protein of 399 amino acids with molecular weight of 43.13 kDa and theoretical isoelectric point of 9.30. Sequence alignment showed that CsPORB was highly homologous to the sequence of POR family protein of other species, indicating conservative evolution of this gene family. The CsPORB expression showed obviously difference in different developmental stages and tissues, and high expression in growing young leaves. Furthermore, both of the PORB and its downstream chlorophyll biosynthesis enzyme activity increased in transgenic 35 S:CsPORB Arabidopsis, as well as chlorophyll content, especially 35 S:CsPORB transgenic plants remained green under dark for one week. Therefore, CsPORB could play an important roles in chlorophyll synthesis pathway of Cymbidium sinense 'Dharma'.
引文
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[15]GABRUK M,STECKA A,STRZA?KA W,et al.Photoactive protochlorophyllide-enzyme complexes reconstituted with PORA,PORBand PORC proteins of A.thaliana:Fluorescence and catalytic properties[J].PLo S One,2015,10(2):e0116990.doi:10.1371/journal.pone.0116990.
[16]LI H M,CULLIGAN K,DIXON R A,et al.CUE1:A mesophyll cellspecific positive regulator of light-controlled gene expression in Arabidopsis[J].Plant Cell,1995,7(10):1599-1610.doi:10.1105/tpc.7.10.1599.
[17]ARMSTRONG G A,RUNGE S,FRICK G,et al.Identification of NADPH:Protochlorophyllide oxidoreductases A and B:A branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana[J].Plant Physiol,1995,108(4):1505-1517.doi:10.1104/pp.108.4.1505.
[18]SAKURABA Y,RAHMAN M L,CHO S H,et al.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions[J].Plant J,2013,74(1):122-133.doi:10.1111/tpj.12110.
[19]RUNGE S,SPERLING U,FRICK G,et al.Distinct roles for lightdependent NADPH:Protochlorophyllide oxidoreductases(POR)A and B during greening in higher plants[J].Plant J,2010,9(4):513-523.doi:10.1046/j.1365-313X.1996.09040513.x.
[20]HOLTORF H,REINBOTHE S,REINBOTHE C,et al.Two routes of chlorophyllide synthesis that are differentially regulated by light in barley(Hordeum vulgare L.)[J].Proc Natl Acad Sci USA,1995,92(8):3254-3258.doi:10.1073/pnas.92.8.3254.
[2]MASUDA T,FUSADA N,OOSAWA N,et al.Functional analysis of isoforms of NADPH:Protochlorophyllide oxidoreductase(POR),PORBand PORC,in Arabidopsis thaliana[J].Plant Cell Physiol,2003,44(10):963-974.doi:10.1093/pcp/pcg128.
[3]REINBOTHE S,REINBOTHE C,LEBEDEV N,et al.PORA and PORB,two light-dependent protochlorophyllide-reducing enzymes of angiosperm chlorophyll biosynthesis[J].Plant Cell,1996,8(5):763-769.doi:10.1105/tpc.8.5.763.
[4]MASUDA T,TAKAMIYA K I.Novel insights into the enzymology,regulation and physiological functions of light-dependent protochlorophyllide oxidoreductase in angiosperms[J].Photosynth Res,2004,81(1):1-29.doi:10.1023/B:PRES.0000028392.80354.7c.
[5]DAHLIN C,ARONSSON H,ALMKVIST J,et al.Protochlorophyllideindependent import of two NADPH:Pchlide oxidoreductase proteins(PORA and PORB)from barley into isolated plastids[J].Physiol Plant,2000,109(3):298-303.doi:10.1034/j.1399-3054.2000.100311.x.
[6]SU Q X,FRICK G,ARMSTRONG G,et al.POR C of Arabidopsis thaliana:A third light-and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light[J].Plant Mol Biol,2001,47(6):805-813.doi:10.1023/A:1013699721301.
[7]FRICK G,SU Q,APEL K,et al.An Arabidopsis porB porC double mutant lacking light-dependent NADPH:Protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrested[J].Plant J,2010,35(2):141-153.doi:10.1046/j.1365-313X.2003.01798.x.
[8]SPERLING U,van CLEVE B,FRICK G,et al.Overexpression of light-dependent PORA or PORB in plants depleted of endogenous PORby far-red light enhances seedling survival in white light and protects against photooxidative damage[J].Plant J,2010,12(3):649-658.doi:10.1046/j.1365-313X.1997.00649.x.
[9]PADDOCK T N,MASON M E,LIMA D F,et al.Arabidopsis protochlorophyllide oxidoreductase A(PORA)restores bulk chlorophyll synthesis and normal development to a porB porC double mutant[J].Plant Mol Biol,2010,72(4/5):445-457.doi:10.1007/s11103-009-9582-y.
[10]WU W J,ELSHEERY N,WEI Q,et al.Defective etioplasts observed in variegation mutants may reveal the light-independent regulation of white/yellow sectors of Arabidopsis leaves[J].J Integr Plant Biol,2011,53(11):846-857.doi:10.1111/j.1744-7909.2011.01079.x.
[11]GAO X L,LI S J,SHAO J F,et al.Preliminary study on isolation,mapping,and function of NADPH:Protochlorophyllide oxidoreductase(POR)gene in rice(Oryza sativa)[J].Plant Physiol J,2015,51(6):860-868.doi:10.13592/j.cnki.ppj.2015.0105.高小丽,李素娟,邵健丰,等.水稻POR基因的分离、定位与功能的初步研究[J].植物生理学报,2015,51(6):860-868.doi:10.13592/j.cnki.ppj.2015.0105.
[12]ZHU G F,YANG F X,SHI S S,et al.Transcriptome characterization of Cymbidium sinense‘Dharma’using 454 pyrosequencing and its application in the identification of genes associated with leaf color variation[J].PLoS One,2015,10(6):e0128592.doi:10.1371/journal.pone.0128592.
[13]XU Q Q,YANG F X,YE Q S,et al.Comparison of photosynthetic pigments content,chlorophyll fluorescence kinetics parameters and chloroplast ultrastructure of leaf mutant cultivars of Cymbidium sinense‘Damo’[J].Chin J Trop Crops,2017,38(7):1210-1215.doi:10.3969/j.issn.1000-2561.2017.07.006.许庆全,杨凤玺,叶庆生,等.墨兰‘达摩’叶艺品系光合色素含量、叶绿素荧光特性和叶绿体超微结构的比较[J].热带作物学报,2017,38(7):1210-1215.doi:10.3969/j.issn.1000-2561.2017.07.006.
[14]JIANG K Y.Cloning and functional analysis of key genes involved in chlorophyll biosynthetic pathway in Pseudosasa japonica cv.Akebobosuji[D].Hangzhou:Zhejiang Agricultural&Forest University,2013:44-55.姜可以.花叶矢竹叶绿素生物合成关键基因PjPORB和PjCAO的克隆与功能分析[D].杭州:浙江农林大学,2013:44-55.
[15]GABRUK M,STECKA A,STRZA?KA W,et al.Photoactive protochlorophyllide-enzyme complexes reconstituted with PORA,PORBand PORC proteins of A.thaliana:Fluorescence and catalytic properties[J].PLo S One,2015,10(2):e0116990.doi:10.1371/journal.pone.0116990.
[16]LI H M,CULLIGAN K,DIXON R A,et al.CUE1:A mesophyll cellspecific positive regulator of light-controlled gene expression in Arabidopsis[J].Plant Cell,1995,7(10):1599-1610.doi:10.1105/tpc.7.10.1599.
[17]ARMSTRONG G A,RUNGE S,FRICK G,et al.Identification of NADPH:Protochlorophyllide oxidoreductases A and B:A branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana[J].Plant Physiol,1995,108(4):1505-1517.doi:10.1104/pp.108.4.1505.
[18]SAKURABA Y,RAHMAN M L,CHO S H,et al.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions[J].Plant J,2013,74(1):122-133.doi:10.1111/tpj.12110.
[19]RUNGE S,SPERLING U,FRICK G,et al.Distinct roles for lightdependent NADPH:Protochlorophyllide oxidoreductases(POR)A and B during greening in higher plants[J].Plant J,2010,9(4):513-523.doi:10.1046/j.1365-313X.1996.09040513.x.
[20]HOLTORF H,REINBOTHE S,REINBOTHE C,et al.Two routes of chlorophyllide synthesis that are differentially regulated by light in barley(Hordeum vulgare L.)[J].Proc Natl Acad Sci USA,1995,92(8):3254-3258.doi:10.1073/pnas.92.8.3254.