黑莓果实发育成熟期木质素合成CAD酶及其基因表达
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摘要
【目的】比较黑莓高硬度品种‘Arapaho’和低硬度品种‘Boysen’果实发育成熟期木质素合成肉桂醇脱氢酶(CAD)基因表达,及其与CAD酶活性及总木质素含量的关系。【方法】对不同发育时期的黑莓果肉,利用半定量RT-PCR分析18个Ru CADs基因成员的表达,利用比色法测定CAD酶活性,利用滴定法测定总木质素含量。【结果】在黑莓的18个Ru CAD基因成员中,有8个CADs基因成员在2个品种发育早、中期果肉中表达量较高;其中Ru CAD16仅在‘Boysen’的早、中期果肉中表达,而在‘Arapaho’中不表达。‘Arapaho’和‘Boysen’果肉CAD酶活性均在着色前即花后9 d和花后21 d时最高,着色后呈下降趋势。‘Arapaho’和‘Boysen’果肉木质素分别在花后24 d和花后27 d含量较高。【结论】‘Arapaho’和‘Boysen’果实着色后,Ru CADs基因成员表达呈多样化,且硬度与Ru CAD酶及木质素含量关系表现出一定差异;而在果实发育后期,Ru CADs基因表达量降低。CAD酶活性降低及木质素含量下降,与黑莓成熟果实硬度下降具一致性,推测木质素合成与成熟果实硬度形成具有相关性。
【Objective】This study aimed to investigate the relationships among the expression profiles of cinnamyl alcohol dehydrogenase( CAD) genes,CAD enzyme activity,and total lignin content in fruit flesh from the fruit-setting to the mature stage of blackberry( Rubus spp.) cv.'Arapaho'and'Boysen',which had relatively high and low firmness,respectively. 【Method】Expression profiles of Ru CAD genes in the fruit flesh of the two blackberry cultivars during the stages from coloring to maturity were detected using semi-quantitative RT-PCR. The changes in CAD enzyme activity and the lignin contents in fruit flesh were determined by colorimetric and titration methods,respectively. 【Result】During fruit development of the two cultivars,eight Ru CAD genes exhibited high expression level in the early and middle stages.Ru CAD16 was only detected in the early and middle stages in'Boysen'fruit flesh and not found in 'Arapaho'during any developmental stage. CAD enzyme activitly level in'Arapaho'and'Boysen'fruits was higher before coloring,specifically 9 DAF( days after flowering) and 21 DAF,and both declined after coloring. Total lignin content in the fruit flesh of both cultivars increased to relatively high level at 24 DAF and 27 DAF,respectively. 【Conclusion】After fruit coloring,expression level of Ru CAD genes exhibited various patterns,and the relationships among the fruit firmness,CAD enzyme activity and lignin content showed differences in fruits of'Arapaho'and'Boysen'. At late developmental stages,the fruit flesh of'Arapaho'and'Boysen'both showed similar declines in the expression level of Ru CAD genes,CAD enzyme activity,and total lignin contents,which was consistent with the decline in blackberry fruit firmness at maturity,implying a relationship between lignin synthesis and firmness in ripe fruits.
【Objective】This study aimed to investigate the relationships among the expression profiles of cinnamyl alcohol dehydrogenase( CAD) genes,CAD enzyme activity,and total lignin content in fruit flesh from the fruit-setting to the mature stage of blackberry( Rubus spp.) cv.'Arapaho'and'Boysen',which had relatively high and low firmness,respectively. 【Method】Expression profiles of Ru CAD genes in the fruit flesh of the two blackberry cultivars during the stages from coloring to maturity were detected using semi-quantitative RT-PCR. The changes in CAD enzyme activity and the lignin contents in fruit flesh were determined by colorimetric and titration methods,respectively. 【Result】During fruit development of the two cultivars,eight Ru CAD genes exhibited high expression level in the early and middle stages.Ru CAD16 was only detected in the early and middle stages in'Boysen'fruit flesh and not found in 'Arapaho'during any developmental stage. CAD enzyme activitly level in'Arapaho'and'Boysen'fruits was higher before coloring,specifically 9 DAF( days after flowering) and 21 DAF,and both declined after coloring. Total lignin content in the fruit flesh of both cultivars increased to relatively high level at 24 DAF and 27 DAF,respectively. 【Conclusion】After fruit coloring,expression level of Ru CAD genes exhibited various patterns,and the relationships among the fruit firmness,CAD enzyme activity and lignin content showed differences in fruits of'Arapaho'and'Boysen'. At late developmental stages,the fruit flesh of'Arapaho'and'Boysen'both showed similar declines in the expression level of Ru CAD genes,CAD enzyme activity,and total lignin contents,which was consistent with the decline in blackberry fruit firmness at maturity,implying a relationship between lignin synthesis and firmness in ripe fruits.
引文
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[3]CAHN H. Fruit quality evaluation of raspberries and blackberries at North Willamette Research and Extension Center[R]. Corvallis or Agricultural Experiment Station Oregon State University,1992.
[4]CHAPLIN G R,BUCKLEY M J,LAI S C. Differential softening and physico-chemical changes in the mesocarp of ripening mango fruit[J]. Acta Horticulturae,1990(269):233-240. DOI:10.17660/Acta Hortic.1990.269.30.
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[8]SALENTIJN E M J,AHARONI A,SCHAART J G,et al. Differential gene expression analysis of strawberry cultivars that differ in fruit-firmness[J]. Physiologia Plantarum,2003,118(4):571-578. DOI:10.1034/j.1399-3054.2003.00138.x.
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[10]KIM S J,KIM M R,BEDGAR D L,et al. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis[J]. Proceedings of the National Academy of Sciences, 2004, 101(6):1455-1460. DOI:10. 1073/pnas.0307987100.
[11]BARAKAT A,BAGNIEWSKA-ZADWORNA A,CHOI A,et al.The cinnamyl alcohol dehydrogenase gene family in Populus:phylogeny,organization,and expression[J]. BMC Plant Biology,2009,9(1):26. DOI:10.1186/1471-2229-9-26.
[12]BURDON J N,SEXTON R. Fruit abscission and ethylene production of four blackberry cultivars(Rubus spp.)[J]. Annals of Applied Biology,2010,123(1):121-132. DOI:10. 1111/j.1744-7348.1993.tb04079.x.
[13]汤飞云,张春红,胡淑英,等.不同品种黑莓果实发育过程中硬度变化规律的调查与分析[J].江西农业学报,2012,24(10):9-11. DOI:10.3969/j.issn.1000-8581.2012.10.003.TANG F Y,ZHANG C H,HU S Y,et al. Changes in fruit firmness of different blackberry cultivars during development and ripening[J]. Acta Agriculturae Jiangxi,2012,24(10):9-11.
[14]LI W L,ZHANG C H,YANG H Y,et al. SSR marker information mining in blackberry from transcriptome sequences[J]. Acta Horticulturae, 2016(1133):97-102. DOI:10. 17660/actahortic.2016.1133.15.
[15]GOFFNER D,JOFFROY I,GRIMA-PETTENATI J,et al. Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem[J]. Planta,1992,188(1):48-53. DOI:10.1007/bf00198938.
[16]SABALLOS A,EJETA G,SANCHEZ E,et al. A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum[Sorghum bicolor(L.)Moench]identifies Sb CAD2 as the brown midrib6 gene[J]. Genetics,2009,181(2):783-795. DOI:10.1534/genetics.108.098996.
[17]祝列克.新世纪中国林木遗传育种发展战略[J].南京林业大学学报,2001,25(1):3-7. DOI:10.3969/j.issn.1000-2006.2001.01.002.ZHU L K. Developmental strategies for forest and tree breeding in China towards to the new century[J]. Journal of Nanjing Forestry University,2001,25(1):3-7.
[18]陈佩珍,吴晓刚,韦蔷,等.松科植物木质素合成相关基因研究进展[J].南京林业大学学报(自然科学版),2017,41(6):169-176. DOI:10.3969/j.issn.1000-2006.201703031.CHEN P Z,WU X G,WEI Q,et al. Research progress of lignin synthesis gene in Pinaceae[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(6):169-176.
[19]吴晓宇,胡尚连,曹颖,等.慈竹CCoAOMT基因的克隆及生物信息学分析[J].南京林业大学学报(自然科学版),2012,36(3):17-22. DOI:10.3969/j.issn.1000-2006.2012.03.005.WU X Y,HU S L,CAO Y,et al. Cloning of CCoAOMT gene in Neosinocalamus affinis and its bioinformatics analysis[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2012,36(3):17-22.
[20] BOERJAN W,RALPH J,BAUCHER M. Lignin biosynthesis[J]. Annual Review of Plant Biology,2003,54(1):519-546.DOI:10.1146/annurev.arplant.54.031902.134938.
[21]李亚东,唐雪东,袁菲,等.我国小浆果生产现状、问题和发展趋势[J].东北农业大学学报,2011,42(1):1-9.LI Y D,TANG X D,YUAN F,et al. Production situation,problems and trends of small fruit in China[J]. Journal of Northeast Agricultural University,2011,42(1):1-9.
[22]张春红,汤飞云,王小敏,等.不同黑莓品种果实形态和硬度分析及其解剖结构和微形态特征观察[J].植物资源与环境学报,2013,22(3):88-94. DOI:10.3969/j.issn.1674-7895.2013.03.14.ZHANG C H,TANG F Y,WANG X M,et al. Analyses on morphology and firmness and observations on anatomical structure and micro-morphological characteristics of fruit of different cultivars of blackberry(Rubus spp.)[J]. Journal of Plant Resources and Environment,2013,22(3):88-94.
[23]PAN H,ZHOU R,LOUIE G V,et al. Structural studies of cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase,key enzymes of monolignol biosynthesis[J]. The Plant Cell,2014,26(9):3709-3727. DOI:10.1105/tpc.114.127399.
[24]鞠志国.采期对莱阳茌梨酚类物质代谢和组织褐变的影响[J].中国农业科学,1991,24(2):63-68.JU Z G. Effects of harvest date on phenolics metabolism and tissue browning of Laiyang Chili(Pyrus bretschneideri Rehd)[J]. Scientia Agricultura Sinica,1991,24(2):63-68.
[25]李红卫,冯双庆.冬枣采后果皮成分及氧化酶活性变化与乙醇积累机理的研究[J].农业工程学报,2003,19(3):165-168. DOI:10.3321/j.issn.1002-6819.2003.03.039.LI H W,FENG S Q. Changes of composition and activity of oxidative enzymes in the pericarp and mechanism of ethanol accumulation in brumal jujube[J]. Transactions of the CSAE,2003,19(3):165-168.
[26]李春燕,张光伦,曾秀丽,等.2006.细胞壁酶活性与甜橙果实质地的相关性研究[J].四川农业大学学报,2006,24(1):73-76. DOI:10.3969/j.issn.1000-2650.2006.01.017.LI C Y,ZHANG G L,ZENG X L,et al. The relationship between the activity of enzyme in cell wall and sweet orange[Citrus sinesis(L.)Osbeck] fruit texture[J]. Journal of Sichuan Agricultural University,2006,24(1):73-76.
[27]BLANCO-ORTALES R,MEDINA-ESCOBAR N,LPEX-Rez J A,et al. Cloning,expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry(Fragaria×ananassa cv.Chandler)[J]. Journal of Experimental Botany, 2002, 53(375):1723-1734. DOI:10. 1093/jxb/erf029.
[28]SHAN L L,LI X,WANG P,et al. Characterization of c DNAs associated with lignification and their expression profiles in loquat fruit with different lignin accumulation[J]. Planta,2008,227(6):1243-1254. DOI:10.1007/s00425-008-0696-2.
[29]吴锦程,唐朝晖,陈群,等.不同贮藏温度对枇杷果肉木质化及相关酶活性的影响[J].武汉植物学研究,2006,24(3):235-239.WU J C,TANG C H,CHEN Q,et al. The effects of different storage temperatures on lignification and related enzymes activities in Loquat pulp[J]. Journal of Wuhan Botanical Research,2006,24(3):235-239.
[2]HARDENBURG R E,WATADA A E,WANG C Y. The commercial storage of fruits,vegetables and florist and nursery stocks[C]//Agriculture Handbook. Washington DC:USDA,1986.
[3]CAHN H. Fruit quality evaluation of raspberries and blackberries at North Willamette Research and Extension Center[R]. Corvallis or Agricultural Experiment Station Oregon State University,1992.
[4]CHAPLIN G R,BUCKLEY M J,LAI S C. Differential softening and physico-chemical changes in the mesocarp of ripening mango fruit[J]. Acta Horticulturae,1990(269):233-240. DOI:10.17660/Acta Hortic.1990.269.30.
[5]WHETTEN R,SEDEROF R. Lignin biosynthesis[J]. The Plant Cell,1995,7(7):1001-1013. DOI:10.1105/tpc.7.7.1001.
[6]VANHOLME R,DEMEDTS B,MORREEL K,et al. Lignin biosynthesis and structure[J]. Plant Physiology,2010,153(3):895-905. DOI:10.1104/pp.110.155119.
[7] BAUCHER M,MONTIES B, MONTAGU M V, et al.Biosynthesis and genetic engineering of lignin[J]. Critical Reviews in Plant Sciences,1998,17(2):125-197. DOI:10.1080/07352689891304203.
[8]SALENTIJN E M J,AHARONI A,SCHAART J G,et al. Differential gene expression analysis of strawberry cultivars that differ in fruit-firmness[J]. Physiologia Plantarum,2003,118(4):571-578. DOI:10.1034/j.1399-3054.2003.00138.x.
[9] ROSATI C,MOURGUES F,GIORNO F,et al. A strawberry EST database for evaluating fruit quality traits and selecting improved genotypes through c DNA microarrays[J]. Acta Horticulturae,2004(663):283-289. DOI:10. 17660/Acta Hortic. 2004.663.46.
[10]KIM S J,KIM M R,BEDGAR D L,et al. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis[J]. Proceedings of the National Academy of Sciences, 2004, 101(6):1455-1460. DOI:10. 1073/pnas.0307987100.
[11]BARAKAT A,BAGNIEWSKA-ZADWORNA A,CHOI A,et al.The cinnamyl alcohol dehydrogenase gene family in Populus:phylogeny,organization,and expression[J]. BMC Plant Biology,2009,9(1):26. DOI:10.1186/1471-2229-9-26.
[12]BURDON J N,SEXTON R. Fruit abscission and ethylene production of four blackberry cultivars(Rubus spp.)[J]. Annals of Applied Biology,2010,123(1):121-132. DOI:10. 1111/j.1744-7348.1993.tb04079.x.
[13]汤飞云,张春红,胡淑英,等.不同品种黑莓果实发育过程中硬度变化规律的调查与分析[J].江西农业学报,2012,24(10):9-11. DOI:10.3969/j.issn.1000-8581.2012.10.003.TANG F Y,ZHANG C H,HU S Y,et al. Changes in fruit firmness of different blackberry cultivars during development and ripening[J]. Acta Agriculturae Jiangxi,2012,24(10):9-11.
[14]LI W L,ZHANG C H,YANG H Y,et al. SSR marker information mining in blackberry from transcriptome sequences[J]. Acta Horticulturae, 2016(1133):97-102. DOI:10. 17660/actahortic.2016.1133.15.
[15]GOFFNER D,JOFFROY I,GRIMA-PETTENATI J,et al. Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem[J]. Planta,1992,188(1):48-53. DOI:10.1007/bf00198938.
[16]SABALLOS A,EJETA G,SANCHEZ E,et al. A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum[Sorghum bicolor(L.)Moench]identifies Sb CAD2 as the brown midrib6 gene[J]. Genetics,2009,181(2):783-795. DOI:10.1534/genetics.108.098996.
[17]祝列克.新世纪中国林木遗传育种发展战略[J].南京林业大学学报,2001,25(1):3-7. DOI:10.3969/j.issn.1000-2006.2001.01.002.ZHU L K. Developmental strategies for forest and tree breeding in China towards to the new century[J]. Journal of Nanjing Forestry University,2001,25(1):3-7.
[18]陈佩珍,吴晓刚,韦蔷,等.松科植物木质素合成相关基因研究进展[J].南京林业大学学报(自然科学版),2017,41(6):169-176. DOI:10.3969/j.issn.1000-2006.201703031.CHEN P Z,WU X G,WEI Q,et al. Research progress of lignin synthesis gene in Pinaceae[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(6):169-176.
[19]吴晓宇,胡尚连,曹颖,等.慈竹CCoAOMT基因的克隆及生物信息学分析[J].南京林业大学学报(自然科学版),2012,36(3):17-22. DOI:10.3969/j.issn.1000-2006.2012.03.005.WU X Y,HU S L,CAO Y,et al. Cloning of CCoAOMT gene in Neosinocalamus affinis and its bioinformatics analysis[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2012,36(3):17-22.
[20] BOERJAN W,RALPH J,BAUCHER M. Lignin biosynthesis[J]. Annual Review of Plant Biology,2003,54(1):519-546.DOI:10.1146/annurev.arplant.54.031902.134938.
[21]李亚东,唐雪东,袁菲,等.我国小浆果生产现状、问题和发展趋势[J].东北农业大学学报,2011,42(1):1-9.LI Y D,TANG X D,YUAN F,et al. Production situation,problems and trends of small fruit in China[J]. Journal of Northeast Agricultural University,2011,42(1):1-9.
[22]张春红,汤飞云,王小敏,等.不同黑莓品种果实形态和硬度分析及其解剖结构和微形态特征观察[J].植物资源与环境学报,2013,22(3):88-94. DOI:10.3969/j.issn.1674-7895.2013.03.14.ZHANG C H,TANG F Y,WANG X M,et al. Analyses on morphology and firmness and observations on anatomical structure and micro-morphological characteristics of fruit of different cultivars of blackberry(Rubus spp.)[J]. Journal of Plant Resources and Environment,2013,22(3):88-94.
[23]PAN H,ZHOU R,LOUIE G V,et al. Structural studies of cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase,key enzymes of monolignol biosynthesis[J]. The Plant Cell,2014,26(9):3709-3727. DOI:10.1105/tpc.114.127399.
[24]鞠志国.采期对莱阳茌梨酚类物质代谢和组织褐变的影响[J].中国农业科学,1991,24(2):63-68.JU Z G. Effects of harvest date on phenolics metabolism and tissue browning of Laiyang Chili(Pyrus bretschneideri Rehd)[J]. Scientia Agricultura Sinica,1991,24(2):63-68.
[25]李红卫,冯双庆.冬枣采后果皮成分及氧化酶活性变化与乙醇积累机理的研究[J].农业工程学报,2003,19(3):165-168. DOI:10.3321/j.issn.1002-6819.2003.03.039.LI H W,FENG S Q. Changes of composition and activity of oxidative enzymes in the pericarp and mechanism of ethanol accumulation in brumal jujube[J]. Transactions of the CSAE,2003,19(3):165-168.
[26]李春燕,张光伦,曾秀丽,等.2006.细胞壁酶活性与甜橙果实质地的相关性研究[J].四川农业大学学报,2006,24(1):73-76. DOI:10.3969/j.issn.1000-2650.2006.01.017.LI C Y,ZHANG G L,ZENG X L,et al. The relationship between the activity of enzyme in cell wall and sweet orange[Citrus sinesis(L.)Osbeck] fruit texture[J]. Journal of Sichuan Agricultural University,2006,24(1):73-76.
[27]BLANCO-ORTALES R,MEDINA-ESCOBAR N,LPEX-Rez J A,et al. Cloning,expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry(Fragaria×ananassa cv.Chandler)[J]. Journal of Experimental Botany, 2002, 53(375):1723-1734. DOI:10. 1093/jxb/erf029.
[28]SHAN L L,LI X,WANG P,et al. Characterization of c DNAs associated with lignification and their expression profiles in loquat fruit with different lignin accumulation[J]. Planta,2008,227(6):1243-1254. DOI:10.1007/s00425-008-0696-2.
[29]吴锦程,唐朝晖,陈群,等.不同贮藏温度对枇杷果肉木质化及相关酶活性的影响[J].武汉植物学研究,2006,24(3):235-239.WU J C,TANG C H,CHEN Q,et al. The effects of different storage temperatures on lignification and related enzymes activities in Loquat pulp[J]. Journal of Wuhan Botanical Research,2006,24(3):235-239.
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