砧木对‘赤霞珠9’葡萄果实发育及隐色花色素还原酶基因表达的影响
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摘要
以‘赤霞珠’嫁接苗和自根苗果实为试材,用Trizol法,对果皮和果肉RNA进行提取和纯化,利用RT-PCR技术对果实隐色花色素还原酶(LAR)转录水平进行研究。结果表明:‘5BB’‘SO4’和‘101-14MG’均增加了‘赤霞珠’葡萄枝条N素质量分数,‘SO4’增加了P素质量分数,‘101-14MG’增加了K素质量分数;‘5BB’和‘SO4’增大了‘赤霞珠’葡萄果粒,‘5BB’使‘赤霞珠’葡萄提前8d转色,‘SO4’拉长了果蒂长度,增加了果穗松散度;在整个‘赤霞珠’果实发育期,各处理果实隐色花色素还原酶活性呈现逐渐下降的趋势,‘101-14MG’和‘SO4’嫁接‘赤霞珠’果实LAR活性始终大于‘赤霞珠’自根苗果实,‘101-14MG’嫁接‘赤霞珠’果实LAR活性最强;果皮和果肉中LAR相对表达量总体呈现下降趋势,果肉中LAR相对表达量极小,果皮中LAR表达量显著高于果肉,‘101-14MG’和‘SO4’嫁接‘赤霞珠’果皮和果肉中LAR相对表达量均大于‘赤霞珠’自根苗和‘5BB’嫁接‘赤霞珠’,其中‘101-14MG’嫁接苗显著大于自根苗。
There are many studies on biosynthesis and accumulation of polyphenols in grape berries under control of environmental conditions.But there are few reports on the effect of rootstock grafting,in particular,the effects of the rootstock on the synthesis and accumulation of LAR have not been reported.In this study,berry of‘Cabernet Sauvignon'grafted and self-rooted as the test material,Trizol method was used to extract and purify RNA from pericarp and pulp.The transcription level of LARin berry was studied by RT-PCR.The results showed that:‘5BB',‘SO4'and‘101-14MG'increased the N mass fraction of the branches of Cabernet Sauvignon,‘SO4'increased the mass fraction of P,‘101-14MG'increased the mass fraction of K;‘5BB'and ‘SO4'increased the volume of‘Cabernet Sauvignon'berries,‘5BB'turned the berries of ‘Cabernet Sauvignon'8days earlier,‘SO4'increased the length of the fruit peduncle and increased the looseness of the cluster.Throughout the development of‘Cabernet Sauvignon',the LAR activity of berries decreased gradually.The LAR activity of Cs/‘SO4'and Cs/‘5BB'berries was always higher than that of ‘Cabernet Sauvignon'rooted shoots,and Cs/‘SO4'berry had the highest LAR activity.The relative expression of LARin the peel and pulp showed a general downward trend,the relative expression of LARin pulp was very low,the expression of LARin pericarp was significantly higher than that in pulp.The relative expression levels of LARin pericarp and berry of Cs/‘SO4'and Cs/‘5BB'were greater than that of‘Cabernet Sauvignon'and Cs/‘101-14MG',with Cs/‘SO4'significantly larger than that of selfrooted shoot.
There are many studies on biosynthesis and accumulation of polyphenols in grape berries under control of environmental conditions.But there are few reports on the effect of rootstock grafting,in particular,the effects of the rootstock on the synthesis and accumulation of LAR have not been reported.In this study,berry of‘Cabernet Sauvignon'grafted and self-rooted as the test material,Trizol method was used to extract and purify RNA from pericarp and pulp.The transcription level of LARin berry was studied by RT-PCR.The results showed that:‘5BB',‘SO4'and‘101-14MG'increased the N mass fraction of the branches of Cabernet Sauvignon,‘SO4'increased the mass fraction of P,‘101-14MG'increased the mass fraction of K;‘5BB'and ‘SO4'increased the volume of‘Cabernet Sauvignon'berries,‘5BB'turned the berries of ‘Cabernet Sauvignon'8days earlier,‘SO4'increased the length of the fruit peduncle and increased the looseness of the cluster.Throughout the development of‘Cabernet Sauvignon',the LAR activity of berries decreased gradually.The LAR activity of Cs/‘SO4'and Cs/‘5BB'berries was always higher than that of ‘Cabernet Sauvignon'rooted shoots,and Cs/‘SO4'berry had the highest LAR activity.The relative expression of LARin the peel and pulp showed a general downward trend,the relative expression of LARin pulp was very low,the expression of LARin pericarp was significantly higher than that in pulp.The relative expression levels of LARin pericarp and berry of Cs/‘SO4'and Cs/‘5BB'were greater than that of‘Cabernet Sauvignon'and Cs/‘101-14MG',with Cs/‘SO4'significantly larger than that of selfrooted shoot.
引文
[1]温鹏飞.葡萄多酚[M].北京:中国农业科学技术出版社,2012:9.WEN P F.Grape Polyphenols[M].Beijing:China Agricultural Science and Technology Press,2012:9.
[2]GARRIDO J,BORGES F.Wine and grape polyphertols:a chemical perspective[J].Food Research International,2011,44(10):3134-3148.
[3]HABRAN A,COMMISSO M,HELWI P,et al.Roostocks/scion/nitrogen interactions affect secondary metabolism in the grape berry[J].Frontiers in Plant Science,2016,7(8):1-11.
[4]骆军,沈国军,王晓庆,等.不同砧木藤稔嫁接苗着色差异及相关酶活性影响[J].上海农业学报,2007,23(3):9-13.LUO J,SHEN G J,WANG X Q,et al.Effects of rootstocks on Fujiminori grape coloration and enzyme activity[J].Acta Agriculturae Shanghai,2007,23(3):9-13.
[5]DIXON R A,XIE D Y,SHARMA S B.Proanthocyanidins-a final frontier in flavonoid research?[J].New Phytologist,2005,165(1):9-28.
[6]MAUGE C,GRANIER T,D'ESTAINTOT B L,et al.Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera[J].Journal of Molecular and Biology,2010,397:1079-1091.
[7]BOGS J,DOWNEY M O,HARVEY J S,et al.Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves[J].Plant Physiology,2005,139:652-663.
[8]阎隆飞,张玉麟.分子生物学[M].北京:北京农业大学出版社,1993.YAN L F,ZHANG Y L.Molecular Biology[M].Beijing:Beijing Agricultural University Press,1993.
[9]QUIROGA A M,DEIS L,CAVAGNARO J B,et al.Water stress and abscisic acid exogenous supply produce differential enhancements in the concentration of selected phenolic compounds in Cabernet Sauvignon[J].Journal of Berry Research,2012(2):33-44.
[10]WEN P F,CHEN J Y,WAN S B,et al.Salicylic acid activates phenylalanine ammonia-lyase in grape berry in response to high temperature stress[J].Plant Growth Regulation,2008,55:1-10.
[11]温鹏飞,邢延富,牛铁泉,等.UV-C对葡萄果实发育过程中黄烷醇类多酚积累及隐色花色素还原酶表达的影响[J].中国农业科学,2012,45(21):4428-4436.WEN P F,XING Y F,NIU T Q,et al.Accumulation of flavanols,expression of leucoanthocyanidin reductase induced by UV-C irradiation during grape berry development[J].Scientia Agricultura Sinica,2012,45(21):4428-4436.
[12]WANG W,TANG K,YANG H R,et al.Distribution of resveratrol and stilbene synthase in young grape plants(Vitis vinifera L.cv.Cabernet Sauvignon)and the effect of UV-C on its accumulation[J].Plant Physiology and Biochemistry,2010,48:142-152.
[13]WANG Y S,GAO L P,SHAN Y,et al.Influence of shade on flavonoid biosynthesis in tea[Camellia sinensis(L.)O.Kuntze][J].Scientia Horticulturae,2012,141:7-16.
[14]OLLE D,GUIRAUD J L,SOUQUET J M,et al.Effect of pre-and postveraison water deficit on proanthocyanidins and anthocyanin accumulation during Shiraz berry development[J].Australian Journal of Grape and Wine Research,2011,17:90-100.
[15]ACEVEDO C,ORTEGA-FARIAS S,FUENTES S.Effects of grapevine(Vitis vinifera L.)water status on water consumption,vegetative growth and grape quality:An irrigation scheduling application to achieve regulated deficit irrigation[J].Agricultural Water Management,2010,97:956-964.
[16]BONGHI C,RIZZINI F M,GAMBUTI A,et al.Phenol compound metabolism and gene expression in the skin of wine grape(Vitis vinifera L.)berries subjected to partial postharvest dehydration[J].Postharvest Biology and Technology,2012,67:102-109.
[17]SANCHEZ E,MORENO D A,FERRERES F,et al.Differential responses of five cherry tomato varieties to water stress:changes on phenolic metabolites and related enzymes[J].Phytochemistry,2011,72:723-729.
[18]温鹏飞,杨运良,高美英,等.干旱对葡萄果实发育过程中黄烷醇类多酚积累及LAR表达的影响[J].园艺学报,2012,39(12):2341-2351.WEN P F,YANG Y L,GAO M Y,et al.The effect of drought on flavanols accumulation and LAR expression during the development of grape berry[J].Acta Horticulturae Sinica,2012,39(12):2341-2351.
[19]温鹏飞.葡萄与葡萄酒中黄烷醇类多酚和果实原花色素合成相关酶表达规律的研究[D].北京:中国农业大学,2005.WEN P F.Studies on flavanols in wine and grape berry and expression of genes involved in proanthocyanidins biosynthesis during berry development[D].Beijing:China Agricultural University,2005.
[20]刘凤之,段长青.葡萄生产配套技术手册[M].北京:中国农业出版社,2012:11.LIU F ZH,DUAN CH Q.Grape Production Supporting Technical Manuals[M].Beijing:China Agricultural Press,2012:11.
[21]温鹏飞.葡萄多酚[M].北京:中国农业科学技术出版社,2012.WEN P F.Grape Polyphenols[M].Beijing:China Agricultural Science and Technology Press,2012.
[22]温鹏飞,牛兴艳,邢延富,等.土壤干旱对葡萄果实中黄烷醇类多酚时空积累及隐色花色素还原酶活性、组织定位的影响[J].中国农业科学,2013,46(14):2979-2989.WEN P F,NIU X Y,XING Y F,et al.Spatial and temporal accumulation of flavanols,activity and tissue localization of leucoanthocyanidin reductase induced by soil drought in developing grape berries[J].Scientia Agricultura Sinica,2013,46(14):2979-2989.
[23]COOKSON S J,MORENO M J C,HEVIN C,et al.Graft union formation in grapevine induces transcriptional changes related to cell wall modification,wounding,hormone signalling,and secondary metabolism[J].Journal of Experimental Botany,2013,12:144.
[24]KIM S A,AHN S Y,YUN H K.Transcriptome analysis of grapevine shoots exposed to chilling temperature for four weeks[J].Horticulture,Environment and Biotechnology,2016,57(2):161-172.
[25]VILVERT E,COSTA M D,CANGAHUALA-INO-CENTE G C,et al.Root proteomic analysis of grapevine rootstocks inoculated with rhizophagus irregularis and Fusarium oxysporumf.sp.herbemontis[J].Revista Brasileira de Ciência do Solo,2017,2:41.
[2]GARRIDO J,BORGES F.Wine and grape polyphertols:a chemical perspective[J].Food Research International,2011,44(10):3134-3148.
[3]HABRAN A,COMMISSO M,HELWI P,et al.Roostocks/scion/nitrogen interactions affect secondary metabolism in the grape berry[J].Frontiers in Plant Science,2016,7(8):1-11.
[4]骆军,沈国军,王晓庆,等.不同砧木藤稔嫁接苗着色差异及相关酶活性影响[J].上海农业学报,2007,23(3):9-13.LUO J,SHEN G J,WANG X Q,et al.Effects of rootstocks on Fujiminori grape coloration and enzyme activity[J].Acta Agriculturae Shanghai,2007,23(3):9-13.
[5]DIXON R A,XIE D Y,SHARMA S B.Proanthocyanidins-a final frontier in flavonoid research?[J].New Phytologist,2005,165(1):9-28.
[6]MAUGE C,GRANIER T,D'ESTAINTOT B L,et al.Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera[J].Journal of Molecular and Biology,2010,397:1079-1091.
[7]BOGS J,DOWNEY M O,HARVEY J S,et al.Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves[J].Plant Physiology,2005,139:652-663.
[8]阎隆飞,张玉麟.分子生物学[M].北京:北京农业大学出版社,1993.YAN L F,ZHANG Y L.Molecular Biology[M].Beijing:Beijing Agricultural University Press,1993.
[9]QUIROGA A M,DEIS L,CAVAGNARO J B,et al.Water stress and abscisic acid exogenous supply produce differential enhancements in the concentration of selected phenolic compounds in Cabernet Sauvignon[J].Journal of Berry Research,2012(2):33-44.
[10]WEN P F,CHEN J Y,WAN S B,et al.Salicylic acid activates phenylalanine ammonia-lyase in grape berry in response to high temperature stress[J].Plant Growth Regulation,2008,55:1-10.
[11]温鹏飞,邢延富,牛铁泉,等.UV-C对葡萄果实发育过程中黄烷醇类多酚积累及隐色花色素还原酶表达的影响[J].中国农业科学,2012,45(21):4428-4436.WEN P F,XING Y F,NIU T Q,et al.Accumulation of flavanols,expression of leucoanthocyanidin reductase induced by UV-C irradiation during grape berry development[J].Scientia Agricultura Sinica,2012,45(21):4428-4436.
[12]WANG W,TANG K,YANG H R,et al.Distribution of resveratrol and stilbene synthase in young grape plants(Vitis vinifera L.cv.Cabernet Sauvignon)and the effect of UV-C on its accumulation[J].Plant Physiology and Biochemistry,2010,48:142-152.
[13]WANG Y S,GAO L P,SHAN Y,et al.Influence of shade on flavonoid biosynthesis in tea[Camellia sinensis(L.)O.Kuntze][J].Scientia Horticulturae,2012,141:7-16.
[14]OLLE D,GUIRAUD J L,SOUQUET J M,et al.Effect of pre-and postveraison water deficit on proanthocyanidins and anthocyanin accumulation during Shiraz berry development[J].Australian Journal of Grape and Wine Research,2011,17:90-100.
[15]ACEVEDO C,ORTEGA-FARIAS S,FUENTES S.Effects of grapevine(Vitis vinifera L.)water status on water consumption,vegetative growth and grape quality:An irrigation scheduling application to achieve regulated deficit irrigation[J].Agricultural Water Management,2010,97:956-964.
[16]BONGHI C,RIZZINI F M,GAMBUTI A,et al.Phenol compound metabolism and gene expression in the skin of wine grape(Vitis vinifera L.)berries subjected to partial postharvest dehydration[J].Postharvest Biology and Technology,2012,67:102-109.
[17]SANCHEZ E,MORENO D A,FERRERES F,et al.Differential responses of five cherry tomato varieties to water stress:changes on phenolic metabolites and related enzymes[J].Phytochemistry,2011,72:723-729.
[18]温鹏飞,杨运良,高美英,等.干旱对葡萄果实发育过程中黄烷醇类多酚积累及LAR表达的影响[J].园艺学报,2012,39(12):2341-2351.WEN P F,YANG Y L,GAO M Y,et al.The effect of drought on flavanols accumulation and LAR expression during the development of grape berry[J].Acta Horticulturae Sinica,2012,39(12):2341-2351.
[19]温鹏飞.葡萄与葡萄酒中黄烷醇类多酚和果实原花色素合成相关酶表达规律的研究[D].北京:中国农业大学,2005.WEN P F.Studies on flavanols in wine and grape berry and expression of genes involved in proanthocyanidins biosynthesis during berry development[D].Beijing:China Agricultural University,2005.
[20]刘凤之,段长青.葡萄生产配套技术手册[M].北京:中国农业出版社,2012:11.LIU F ZH,DUAN CH Q.Grape Production Supporting Technical Manuals[M].Beijing:China Agricultural Press,2012:11.
[21]温鹏飞.葡萄多酚[M].北京:中国农业科学技术出版社,2012.WEN P F.Grape Polyphenols[M].Beijing:China Agricultural Science and Technology Press,2012.
[22]温鹏飞,牛兴艳,邢延富,等.土壤干旱对葡萄果实中黄烷醇类多酚时空积累及隐色花色素还原酶活性、组织定位的影响[J].中国农业科学,2013,46(14):2979-2989.WEN P F,NIU X Y,XING Y F,et al.Spatial and temporal accumulation of flavanols,activity and tissue localization of leucoanthocyanidin reductase induced by soil drought in developing grape berries[J].Scientia Agricultura Sinica,2013,46(14):2979-2989.
[23]COOKSON S J,MORENO M J C,HEVIN C,et al.Graft union formation in grapevine induces transcriptional changes related to cell wall modification,wounding,hormone signalling,and secondary metabolism[J].Journal of Experimental Botany,2013,12:144.
[24]KIM S A,AHN S Y,YUN H K.Transcriptome analysis of grapevine shoots exposed to chilling temperature for four weeks[J].Horticulture,Environment and Biotechnology,2016,57(2):161-172.
[25]VILVERT E,COSTA M D,CANGAHUALA-INO-CENTE G C,et al.Root proteomic analysis of grapevine rootstocks inoculated with rhizophagus irregularis and Fusarium oxysporumf.sp.herbemontis[J].Revista Brasileira de Ciência do Solo,2017,2:41.