干扰LYC-B基因调控番茄果实挥发性物质及主要品质性状
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摘要
构建含有番茄红素β-环化酶(lycopeneβ-cyclase,LYC-B)基因LYC-B中801 bp和301 bp两段保守序列的RNA干扰植物表达载体,农杆菌果实注射法遗传转化紫色番茄果实,通过卡那霉素抗性筛选、聚合酶链式反应及LYC-B基因表达量鉴定获得转基因植株,并对转基因植株成熟果实的挥发性物质及主要品质性状进行测定。结果表明,干扰LYC-B基因后番茄果实中检测到的挥发性物质主要为醇类、醛类、酮类、酯类和其他类。与野生型相比,转基因植株中总挥发性物质种类增加了6种:醛类3种、酮类2种、酯类1种;醇类个数不变;其他类减少1种。醛类物质相对含量不变,醇类物质相对含量减少,酮类、酯类和其他类相对含量增加。特征挥发性物质反-2-庚烯醛、6-甲基-5-庚烯-2-酮、水杨酸甲酯和2-异丁基噻唑等的相对含量增加。番茄红素的含量增加了16.78%,可溶性固形物、抗坏血酸和总酸等含量增加,β-胡萝卜素和总糖含量变化显著。干扰LYC-B基因不仅影响了番茄果实的挥发性物质,还增加了番茄红素、可溶性固形物、抗坏血酸、总酸等的含量。
This work aimed to regulate the volatile components and major quality traits of tomato fruits by down-regulation of the expression of lycopene β-cyclase(LYC-B) gene using antisense RNA interference technique.Purple tomato fruits were transformed by injection of the plant expression vector containing two conserved sequences(801 and 301 bp,respectively)of LYC-B gene.The transgenic plants were obtained kanamycin resistance screening,polymerase chain reaction and quantification of LYC-B gene expression.The volatile substances and major quality traits of ripe tomato fruits were compared between the transgenic and wild-type(WT) plants.The results showed that alcohols,aldehydes,ketones,esters and other volatiles were detected in transgenic tomato fruits.Compared with the wild-type control,six new volatile compounds were identified from the transgenic fruits,consisting of three aldehydes,two ketones,and one ester,and one compound belonging to another chemical class was detected in the control rather than the transgenic fruits.In addition,the same relative content of aldehydes was observed between both fruits,but the transgenic fruits showed decreased relative content of alcohols and increased relative contents of ketones,esters and other compounds.The characteristic volatile compounds were also increased,such as(E) 2-heptenal,6-methyl 5-hepten-2-one,methyl salicylate and 2-isobutylthiazole,and lycopene increase by 16.78%compared with the wild-type fruits.In addition,the contents of soluble solids,ascorbic acid and total acid were also increased.On the other hand,the contents of β-carotene and total sugar were identical to those of the WT fruits.Therefore,down-regulated expression of LYC-B gene by RNA interference can affect volatile substances and enhance the contents of lycopene,soluble solids,ascorbic acid and total acid,thereby improving the nutritional quality of tomato fruits.
This work aimed to regulate the volatile components and major quality traits of tomato fruits by down-regulation of the expression of lycopene β-cyclase(LYC-B) gene using antisense RNA interference technique.Purple tomato fruits were transformed by injection of the plant expression vector containing two conserved sequences(801 and 301 bp,respectively)of LYC-B gene.The transgenic plants were obtained kanamycin resistance screening,polymerase chain reaction and quantification of LYC-B gene expression.The volatile substances and major quality traits of ripe tomato fruits were compared between the transgenic and wild-type(WT) plants.The results showed that alcohols,aldehydes,ketones,esters and other volatiles were detected in transgenic tomato fruits.Compared with the wild-type control,six new volatile compounds were identified from the transgenic fruits,consisting of three aldehydes,two ketones,and one ester,and one compound belonging to another chemical class was detected in the control rather than the transgenic fruits.In addition,the same relative content of aldehydes was observed between both fruits,but the transgenic fruits showed decreased relative content of alcohols and increased relative contents of ketones,esters and other compounds.The characteristic volatile compounds were also increased,such as(E) 2-heptenal,6-methyl 5-hepten-2-one,methyl salicylate and 2-isobutylthiazole,and lycopene increase by 16.78%compared with the wild-type fruits.In addition,the contents of soluble solids,ascorbic acid and total acid were also increased.On the other hand,the contents of β-carotene and total sugar were identical to those of the WT fruits.Therefore,down-regulated expression of LYC-B gene by RNA interference can affect volatile substances and enhance the contents of lycopene,soluble solids,ascorbic acid and total acid,thereby improving the nutritional quality of tomato fruits.
引文
[1]刘晓英,常涛涛,郭世荣,等.红蓝LED光全生育期照射对樱桃番茄果实品质的影响[J].中国蔬菜,2010,30(22):21-27.
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[11]王巧丽.番茄红素-β-环化酶基因干扰载体的构建及其对不同果色番茄注射转化的研究[D].杨凌:西北农林科技大学,2014:15-16.
[12]李翠.番茄SpMPKs基因响应非生物胁迫的功能分析[D].杨凌:西北农林科技大学,2014:10-12.
[13]VILJANEN K,LILLE M,HEINIO R L,et al.Effect of high-pressure processing on volatile composition and odour of cherry tomato puree[J].Food Chemistry,2011,129(4):1759-1765.DOI:10.1016/j.foodchem.2011.06.046.
[14]常培培,梁燕,张静,等.5种不同果色樱桃番茄品种果实挥发性物质及品质特性分析[J].食品科学,2014,35(22):215-221.DOI:10.7506/spkx1002-6630-201422042.
[15]唐晓伟,刘明池,何洪巨.新鲜番茄风味组分研究[J].食品科学,2007,28(2):28-30.DOI:10.3321/j.issn:1002-6630.2007.02.002.
[16]WANG L B,BALDWIN E A,PLOTTO A,et al.Effect of methyl salicylate and methyl jasmonate pre-treatment on the volatile profile in tomato fruit subjected to chilling temperature[J].Postharvest Biology and Technology,2015,108:28-38.DOI:10.1016/j.postharvbio.2015.05.005.
[17]甘霖,申琳,生吉萍.秸秆源品质改良因子采前处理对番茄果实品质的影响[J].食品科学,2013,34(4):221-225.
[18]陈书霞,林海军.番茄果实不同发育阶段香气成分组成及变化[J].西北植物学报,2010,30(11):2258-2264.
[19]张志良,翟伟,李小方.植物生理学实验指导[M].4版.北京:北京高等教育出版社,2010:100-112.
[20]刘沐霖,惠伯棣,庞克诺.番茄及其制品中番茄红素含量的C_(18)-HPLC-PDA定量分析[J].食品科学,2007,28(7):453-456.
[21]刘明池,郝静,唐晓伟.番茄果实芳香物质的研究进展[J].中国农业科学,2008,41(5):1444-1451.
[22]徐苏萌,宋焕禄,高艳明,等.不同基质配比对番茄风味成分的影响[J].湖北农业科学,2015,54(15):3689-3691.DOI:10.14088/j.cnki.issn0439-8114.2015.15.027.
[23]TIEMAN D,BLISS P,MCINTYRE L M,et al.The chemical interactions underlying tomato flavor preferences[J].Current Biology,2012,22(11):1035-1039.DOI:10.1016/j.cub.2012.04.016.
[24]郭东花,赵彩萍,张静,等.不同钾肥用量对“重阳红”桃果实挥发性物质的影响[J].食品科学,2016,37(2):109-114.DOI:10.7506/spkx1002-6630-201602019.
[25]SOCACI S A,SOCACIU C,MURESAN C,et al.Chemometric discrimination of different tomato cultivars based on their volatile fingerprint in relation to lycopene and total phenolics content[J].Phytochemical Analysis,2014,25(2):161-169.DOI:10.1002/pca.2483.
[26]顾胜华,李湘洲,张盛伟.β-紫罗兰酮合成的研究进展[J].中国食品添加剂,2015(4):173-178.DOI:10.3969/j.issn.1006-2513.2015.04.025.
[27]CHAMBERS E,KOPPEL K.Associations of volatile compounds with sensory aroma and flavor:the complex nature of flavor[J].Molecules,2013,18(5):4887-4905.DOI:10.3390/molecules18054887.
[2]VOGEL J T,TIEMAN D M,SIMS C A,et al.Carotenoid content impacts flavor acceptability in tomato(Solanum lycopersicum)[J].Journal of the Science of Food and Agriculture,2010,90(13):2233-2240.DOI:10.1002/jsfa.4076.
[3]朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理与分子生物学学报,2004,30(6):609-618.DOI:10.3321/j.issn:1671-3877.2004.06.002.
[4]徐加新,梁燕.LYC-b基因反义表达对番茄果实番茄红素含量的影响[J].西北农林科技大学学报(自然科学版),2009,37(12):127-132.
[5]吴江敏,孙亚东,梁燕,等.番茄红素-β-环化酶(LYC-b)反义基因在番茄转基因后代的表达及其遗传稳定性[J].农业生物技术学报,2011,19(5):801-807.DOI:10.3969/j.issn.1674-7968.2011.05.003.
[6]阮美颖,叶青静,周国治,等.不同颜色樱桃番茄果实成熟过程中色素的变化[J].浙江农业科学,2013,5(5):526-528.DOI:10.3969/j.issn.0528-9017.2013.05.013.
[7]吕洁,梁燕,赵菁菁,等.LYC-B基因沉默对紫色番茄果实主要色素及挥发性物质的影响[J].食品科学,2015,36(23):221-227.DOI:10.7506/spkx1002-6630-201523041.
[8]王巧丽,梁燕,张振才,等.番茄果实特异性LYC-B干扰载体的构建及在不同颜色果实中的表达特异性验证[J].中国蔬菜,2014,5(5):11-18.DOI:10.3969/j.issn.1000-6346.2014.05.003.
[9]YASMEEN A,MIRZA B,IN AYATULLAH S,et al.In planta transformation of tomato[J].Plant Molecular Biology Reporter,2009,27(1):20-28.DOI:10.1007/s11105-008-0044-5.
[10]BALDWIN E A,SCOTT J W,SHEWMAKER C K,et al.Flavor trivia and tomato aroma:biochemistry and possible mechanisms for control of important aroma components[J].HortScience,2000,35(6):1013-1022.DOI:10.1002/pca.2483.
[11]王巧丽.番茄红素-β-环化酶基因干扰载体的构建及其对不同果色番茄注射转化的研究[D].杨凌:西北农林科技大学,2014:15-16.
[12]李翠.番茄SpMPKs基因响应非生物胁迫的功能分析[D].杨凌:西北农林科技大学,2014:10-12.
[13]VILJANEN K,LILLE M,HEINIO R L,et al.Effect of high-pressure processing on volatile composition and odour of cherry tomato puree[J].Food Chemistry,2011,129(4):1759-1765.DOI:10.1016/j.foodchem.2011.06.046.
[14]常培培,梁燕,张静,等.5种不同果色樱桃番茄品种果实挥发性物质及品质特性分析[J].食品科学,2014,35(22):215-221.DOI:10.7506/spkx1002-6630-201422042.
[15]唐晓伟,刘明池,何洪巨.新鲜番茄风味组分研究[J].食品科学,2007,28(2):28-30.DOI:10.3321/j.issn:1002-6630.2007.02.002.
[16]WANG L B,BALDWIN E A,PLOTTO A,et al.Effect of methyl salicylate and methyl jasmonate pre-treatment on the volatile profile in tomato fruit subjected to chilling temperature[J].Postharvest Biology and Technology,2015,108:28-38.DOI:10.1016/j.postharvbio.2015.05.005.
[17]甘霖,申琳,生吉萍.秸秆源品质改良因子采前处理对番茄果实品质的影响[J].食品科学,2013,34(4):221-225.
[18]陈书霞,林海军.番茄果实不同发育阶段香气成分组成及变化[J].西北植物学报,2010,30(11):2258-2264.
[19]张志良,翟伟,李小方.植物生理学实验指导[M].4版.北京:北京高等教育出版社,2010:100-112.
[20]刘沐霖,惠伯棣,庞克诺.番茄及其制品中番茄红素含量的C_(18)-HPLC-PDA定量分析[J].食品科学,2007,28(7):453-456.
[21]刘明池,郝静,唐晓伟.番茄果实芳香物质的研究进展[J].中国农业科学,2008,41(5):1444-1451.
[22]徐苏萌,宋焕禄,高艳明,等.不同基质配比对番茄风味成分的影响[J].湖北农业科学,2015,54(15):3689-3691.DOI:10.14088/j.cnki.issn0439-8114.2015.15.027.
[23]TIEMAN D,BLISS P,MCINTYRE L M,et al.The chemical interactions underlying tomato flavor preferences[J].Current Biology,2012,22(11):1035-1039.DOI:10.1016/j.cub.2012.04.016.
[24]郭东花,赵彩萍,张静,等.不同钾肥用量对“重阳红”桃果实挥发性物质的影响[J].食品科学,2016,37(2):109-114.DOI:10.7506/spkx1002-6630-201602019.
[25]SOCACI S A,SOCACIU C,MURESAN C,et al.Chemometric discrimination of different tomato cultivars based on their volatile fingerprint in relation to lycopene and total phenolics content[J].Phytochemical Analysis,2014,25(2):161-169.DOI:10.1002/pca.2483.
[26]顾胜华,李湘洲,张盛伟.β-紫罗兰酮合成的研究进展[J].中国食品添加剂,2015(4):173-178.DOI:10.3969/j.issn.1006-2513.2015.04.025.
[27]CHAMBERS E,KOPPEL K.Associations of volatile compounds with sensory aroma and flavor:the complex nature of flavor[J].Molecules,2013,18(5):4887-4905.DOI:10.3390/molecules18054887.