葡萄CO4基因的克隆及其对光质响应的表达分析
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摘要
为了探究不同光质以及转光条件对葡萄CO4基因表达水平及试管苗叶绿素荧光参数的影响,利用RT-PCR技术,从葡萄贝达试管苗中克隆了一个光质响应基因CO4(Gen Bank登录号为KY652090),并对其进行生物信息学以及q RT-PCR分析。结果表明,CO4基因片段全长663 bp,编码了248个氨基酸,其开放阅读框为747 bp;葡萄CO4有4个跨膜区,预测其为跨膜蛋白;葡萄CO4的分子式为C_(1253)H_(1990)N_(324)O_(340)S_(11),预测是疏水性不稳定蛋白。系统进化树分析表明,该蛋白与巨桉CO4最为相似。经荧光实时定量PCR分析表明,在红光转蓝光处理下葡萄CO4表达量显著高于白光(对照),是对照的5. 39倍;其次为蓝光处理;在白光转蓝光条件下CO4表达量最低,是对照的54%;红白转光处理下CO4表达量与对照无显著差异。叶绿素荧光参数分析结果显示,红光转蓝光处理后的Fv/Fm(最大光化学效率)、Fv/Fo(潜在活性)和q P(光化学猝灭系数)均显著高于对照,NPQ在该处理下(非光化学猝灭系数)最低;在红光处理下NPQ最高,Fv/Fm、q P最低。葡萄CO4对光质敏感,且在不同光质及转光条件下其响应水平不同,红光转蓝光处理下上调表达显著,且该光照条件下能显著促进葡萄试管苗的光化学能力。为深入研究CO基因在葡萄试管苗感光机理中的作用提供理论依据,并为葡萄光质改良奠定基础。
To explore the effects of different light qualities on the expression of CO4 gene in grapevine plantlet in vitro. The full-length cDNA sequence of CO4 was cloned from Vitis riparia × V. labrusca Beta in vitro by RTPCR. Bioinformatics analysis of the second level structure,and phylogenetic tree of protein. The result showed that the size of CO4 gene fragment was 663 bp,the open reading frame( ORF) of CO4 gene was 747 bp,including 248 amino acid coding regions. By use bioinformation analysis,CO4 of Beta was a hydrophobic,unstable protein with good lipid solubility. CO4 had a signal peptide,it can be inferred that it was a transmembrane protein. Phylogenetic tree indicated that CO4 of Beta( Vitis riparia × V. labrusca) had the highest evolutionary relationship with Eucalyptus grandis( Eucalyptus grandis Hill ex Maiden). qRT-PCR analysis showed that the CO4 expressed specifically and strongly in the treatment of red to blue light( control),which increased 5. 39 fold in comparison to the control. The difference was not significant when exposed to the treatment of white to red light and red to white light. However the expression of CO4 was the lowest in the white to blue light treatment,which was 54% of the control. Fluorescence parameter analysis showed that the maximum photochemical efficiency( Fv/Fm),potential activity( Fv/Fo) and photochemical quenching coefficient( qP) were significantly higher than those of the control under red to blue light. The non photochemical quenching coefficient( NPQ) was the lowest under that treatment,but was the highest under red light treatment,it was the lowest under this treatment. The CO4 of Beta was more sensitive to light. The treatmentsof red to blue light could not only promote the expression of CO4,but also could improve the Beta Grapevine in vitro photosynthetic capacity. Thus,red to blue light played an important role in the process of morphogenesis of Beta Grapevine in vitro.
To explore the effects of different light qualities on the expression of CO4 gene in grapevine plantlet in vitro. The full-length cDNA sequence of CO4 was cloned from Vitis riparia × V. labrusca Beta in vitro by RTPCR. Bioinformatics analysis of the second level structure,and phylogenetic tree of protein. The result showed that the size of CO4 gene fragment was 663 bp,the open reading frame( ORF) of CO4 gene was 747 bp,including 248 amino acid coding regions. By use bioinformation analysis,CO4 of Beta was a hydrophobic,unstable protein with good lipid solubility. CO4 had a signal peptide,it can be inferred that it was a transmembrane protein. Phylogenetic tree indicated that CO4 of Beta( Vitis riparia × V. labrusca) had the highest evolutionary relationship with Eucalyptus grandis( Eucalyptus grandis Hill ex Maiden). qRT-PCR analysis showed that the CO4 expressed specifically and strongly in the treatment of red to blue light( control),which increased 5. 39 fold in comparison to the control. The difference was not significant when exposed to the treatment of white to red light and red to white light. However the expression of CO4 was the lowest in the white to blue light treatment,which was 54% of the control. Fluorescence parameter analysis showed that the maximum photochemical efficiency( Fv/Fm),potential activity( Fv/Fo) and photochemical quenching coefficient( qP) were significantly higher than those of the control under red to blue light. The non photochemical quenching coefficient( NPQ) was the lowest under that treatment,but was the highest under red light treatment,it was the lowest under this treatment. The CO4 of Beta was more sensitive to light. The treatmentsof red to blue light could not only promote the expression of CO4,but also could improve the Beta Grapevine in vitro photosynthetic capacity. Thus,red to blue light played an important role in the process of morphogenesis of Beta Grapevine in vitro.
引文
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[16] Almada R,Cabrera N,Casaretto J A,et al. Vv CO and Vv COL1,two CONSTANS homologous genes,are regulated during flower induction and dormancy in grapevine buds[J]. Plant Cell Reports,2009,28(8):1193-1203.
[17] Chaurasia A K,Patil H B,Azeez A,et al. Molecular characterization of CONSTANS-Like(COL)genes in banana(Musa acuminata L. AAA Group,cv. Grand Nain)[J]. Physiol Mol Biol Plants,2016,22(1):1-15.
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[20] Fu J,Yang L,Dai S. Identification and characterization of the CONSTANS-like gene family in the short-day plant Chrysanthemum lavandulifolium[J]. Molecular Genetics&Genomics,2015,290(3):1039-1054.
[21]马宗桓,陈佰鸿,毛娟,等.人参果长丽叶片再生体系的建立[J].中国农学通报,2014,30(22):197-202.
[22]马宗桓,毛娟,李文芳,等.葡萄Sn RK2家族基因的鉴定与表达分析[J].园艺学报,2016,43(10):1891-1903.
[23] Valverde F. CONSTANS and the evolutionary origin of photoperiodic timing of flowering[J]. Journal of Experimental Botany,2011,62(8):2453-2463.
[24] Tan J,Wu F,Wan J. Flowering time regulation by the CONSTANS-Like gene Os COL10[J]. Plant Signaling&Behavior,2017,12(1):e1267893.
[25]曾幼玲,杨瑞瑞.植物mi RNA的生物学特性及在环境胁迫中的作用[J].中国农业科学,2016,49(19):3671-3682.
[26] Imaizumi T,Schultz T F,Harmon F G,et al. FKF1FBOX protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J]. Science,2005,309(5732):293-297.
[27]樊丽娜,邓海华,齐永文.植物CO基因研究进展[J].西北植物学报,2008,28(6):1281-1287.
[28] Sawa M,Nusinow D A,Kay S A,et al. FKF1 and GIGANTEA complex formation is required for daylength measurement in Arabidopsis[J]. Science,2007,318(5848):261-265.
[29] Bondada B R,Syvertsen J P. Leaf chlorophyⅡ,net gas exchange and chloroplast ultrastructure in citrus leaves of different nitrogen status[J]. Tree Physiology,2003,23(8):553-559.
[30]张文林,任亚超,张益文,等.不同光质LED光源对转基因杨树叶片Bt毒蛋白含量及叶绿素荧光参数的影响[J].核农学报,2016,30(8):1639-1645.
[31] Franklin K A,Quail P H. Phytochrome functions in Arabidopsis development[J]. Journal of Experimental Botany,2010,61(1):11-24.
[2] Sindelar T J,Millar K D,Kiss J Z. Red light effects on blue light-based phototropism in roots of Arabidopsis thaliana[J]. International Journal of Plant Sciences,2014,175(6):731-740.
[3] Guo F,Wang C,Liu H P,et al. Effect of light quality and light intensity on the chlorophyⅡfluorescence parameters of red raspberry tissue culture plantlets[J]. Northern Horticulture,2016(11):27-31.
[4] Ouzounis T,Heuvelink E,Ji Y,et al. Blue and red led lighting effects on plant biomass,stomatal conductance,and metabolite content in nine tomato genotypes[J]. Acta Horticulturae,2016,1134(1134):251-258.
[5] Samuoliene G,Sirtautas R,Brazaityte A A,et al. Supplementary red-LED lighting and the changes in phytochemical content of two baby leaf lettuce varieties during three seasons[J]. Journal of Food Agriculture&Environment,2012,10(3/4,2):701-706.
[6] Alvarenga I A,Pacheco F V,Silca S T,et al. In vitro culture of Achillea millefolium L.:quality and intensity of light on growth and production of volatiles[J]. Plant Cell Tissue&Organ Culture,2015,122(2):299-308.
[7] Cameron R F,Harrisonmurray R S,Judd H L,et al. The effects of photoperiod and light spectrum on stock plant growth and rooting of cuttings of Cotinus coggygria'Royal Purple'[J]. Journal of Horticultural Science&Biotechnology,2015,80(2):245-253.
[8] Liang Z S,Li Q,Xu W H. Effects of different light quality on growth,active ingredients and enzymes activities of Salvia miltiorrhiza[J]. China Journal of Chinese Materia Medica,2012,37(14):2055-2060.
[9]刘媛,李胜,马绍英,等.不同光质对葡萄试管苗离体培养生长发育的影响[J].园艺学报,2009,36(8):1105-1112.
[10] Sindelar T J,Millar K L,Kiss J Z. Red light effects on blue light-based phototropism in roots of Arabidopsis thaliana[J]. International Journal of Plant Sciences,2014,175(6):731-740.
[11] Sun W,Ubierna N,Ma J Y,et al. The coordination of C4photosynthesis and the CO2-concentrating mechanism in maize and Miscanthus×giganteus in response to transient changes in light quality[J]. Plant Physiology,2014,164(3):1283-1292.
[12] Wong A C S,Hecht V F G,Picard K,et al. Isolation and functional analysis of CONSTANS-LIKE genes suggests that a central role for CONSTANS in flowering time control is not evolutionarily conserved in Medicago truncatula[J]. Frontiers in Plant Science,2014,5(17):486-486.
[13]冉从福,邵慧,余静,等.小麦CO-like基因TaCO9的克隆及分析[J].麦类作物学报,2014,34(10):1319-1326.
[14] Drabe2ováJ,Cháb D,Kolar^J,et al. A dark-light transition triggers expression of the floral promoter Cr FTL1and downregulates CONSTANS-like genes in a short-day plant Chenopodium rubrum[J]. Journal of Experimental Botany,2014,65(8):2137.
[15]叶雪凌,刘志勇,王孝宣,等.番茄CONSTANS类似基因的克隆与表达分析[J].园艺学报,2008,35(11):1607-1612.
[16] Almada R,Cabrera N,Casaretto J A,et al. Vv CO and Vv COL1,two CONSTANS homologous genes,are regulated during flower induction and dormancy in grapevine buds[J]. Plant Cell Reports,2009,28(8):1193-1203.
[17] Chaurasia A K,Patil H B,Azeez A,et al. Molecular characterization of CONSTANS-Like(COL)genes in banana(Musa acuminata L. AAA Group,cv. Grand Nain)[J]. Physiol Mol Biol Plants,2016,22(1):1-15.
[18] Datta S,Hettiarachchi G H,Deng X W,et al. Arabidop-sis CONSTANS-LIKE 3 is a positive regulator of red light signaling and root growth[J]. The Plant Cell,2006,18(1):70-84.
[19] Hassidim M,Harir Y,Yakir E,et al. Over-expression of CONSTANS-LIKE 5 can induce flowering in short-day grown Arabidopsis[J]. Planta,2009,230(3):481-491.
[20] Fu J,Yang L,Dai S. Identification and characterization of the CONSTANS-like gene family in the short-day plant Chrysanthemum lavandulifolium[J]. Molecular Genetics&Genomics,2015,290(3):1039-1054.
[21]马宗桓,陈佰鸿,毛娟,等.人参果长丽叶片再生体系的建立[J].中国农学通报,2014,30(22):197-202.
[22]马宗桓,毛娟,李文芳,等.葡萄Sn RK2家族基因的鉴定与表达分析[J].园艺学报,2016,43(10):1891-1903.
[23] Valverde F. CONSTANS and the evolutionary origin of photoperiodic timing of flowering[J]. Journal of Experimental Botany,2011,62(8):2453-2463.
[24] Tan J,Wu F,Wan J. Flowering time regulation by the CONSTANS-Like gene Os COL10[J]. Plant Signaling&Behavior,2017,12(1):e1267893.
[25]曾幼玲,杨瑞瑞.植物mi RNA的生物学特性及在环境胁迫中的作用[J].中国农业科学,2016,49(19):3671-3682.
[26] Imaizumi T,Schultz T F,Harmon F G,et al. FKF1FBOX protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J]. Science,2005,309(5732):293-297.
[27]樊丽娜,邓海华,齐永文.植物CO基因研究进展[J].西北植物学报,2008,28(6):1281-1287.
[28] Sawa M,Nusinow D A,Kay S A,et al. FKF1 and GIGANTEA complex formation is required for daylength measurement in Arabidopsis[J]. Science,2007,318(5848):261-265.
[29] Bondada B R,Syvertsen J P. Leaf chlorophyⅡ,net gas exchange and chloroplast ultrastructure in citrus leaves of different nitrogen status[J]. Tree Physiology,2003,23(8):553-559.
[30]张文林,任亚超,张益文,等.不同光质LED光源对转基因杨树叶片Bt毒蛋白含量及叶绿素荧光参数的影响[J].核农学报,2016,30(8):1639-1645.
[31] Franklin K A,Quail P H. Phytochrome functions in Arabidopsis development[J]. Journal of Experimental Botany,2010,61(1):11-24.