苹果苦痘病对果实类黄酮总含量的影响及其分子生物学机制
详细信息    查看全文 | 推荐本文 | 收藏本文
  • 英文篇名:Influence of bitter pit on the flavonoid contents in apple fruits and its molecular biological mechanism revealed by the whole-transcriptome RNA-Seq analysis
  • 作者:余贤美 ; 薛晓敏 ; 王金政 ; 王贵平 ; 聂佩显 ; 陈汝 ; 韩雪平
  • 英文作者:Xianmei Yu;Xiaomin Xue;Jinzheng Wang;Guiping Wang;Peixian Nie;Ru Chen;Xueping Han;Shandong Institute of Pomology;
  • 关键词:全转录组测序分析 ; 苹果苦痘病 ; qRT-PCR分析 ; 类黄酮生物合成与积累 ; 类黄酮总含量
  • 英文关键词:transcriptome sequencing analysis;;bitter pit in apples;;qRT-PCR analysis;;flavonoid biosynthesis and accumulation;;the total flavonoid content
  • 中文刊名:科学通报
  • 英文刊名:Chinese Science Bulletin
  • 机构:山东省果树研究所;
  • 出版日期:2019-06-30
  • 年:2019
  • 期:18
  • 基金:国家现代农业产业技术体系(CARS-27);; 国家重点研发计划(2014BAD16B02-2);; 山东省重点研发计划(2017CXGC0210);; 国家自然科学基金(31600021)资助
  • 页:44-56
  • CN:11-1784/N
  • ISSN:0023-074X
  • 分类号:S436.611.1
摘要
类黄酮在多种植物生物学功能和人类慢性病的治疗上发挥了重要的作用.苹果苦痘病是苹果生产上的一种重要生理性病害,严重影响了果品的品质,造成了巨大经济损失.研究苹果苦痘病对苹果果实类黄酮的影响及其分子生物学机制,可为医用类黄酮的获得挖掘新的资源.采用氯化铝比色法测定了健康果实和苦痘病果的类黄酮总含量,结果显示,苦痘病果的类黄酮总含量为健康果实的4.28倍.通过转录组测序和qRT-PCR对类黄酮总含量变化的分子生物学机制进行了研究.经测序,从苹果健康果实和苦痘病果2组样品中,分别获得226.702和202.951 M Clean reads,包含32.807和29.626 Gb测序数据,其碱基百分比(Q30)大于95.0%.以健康果实为参考,苦痘病果共获得2632个差异表达基因,其中上调基因2080个,下调基因552个.经GO基因功能注释和KEGG代谢通路富集分析,获得与类黄酮合成相关的差异表达基因26个(24个上调基因和2个下调基因),选取其中8个差异表达基因:CYP98A3(1), CYP98A3 (1), BADH, DAT, MdHCT (1), MdHCT (2), CHI (1)和CHI (2)进行qRT-PCR分析验证,结果显示,该8个基因均表现为上调表达,上调倍数为1.05~7.17倍.研究表明,苹果苦痘病的发生刺激了苹果果实中类黄酮的大量表达和积累,可为医学研究中类黄酮的获得,提供更广泛的资源.
    Flavonoids are an important group of polyphenolic compounds which are widely distributed throughout the plant kingdom especially in colored fruits and flowers. They play important roles in many plant biological functions and have therapeutic effect on many chronic diseases in humans. The flavonoid biosynthesis and productivity in plants could be stimulated by many biotic and abiotic factors. Bitter pit in apple is an important physiological disorder in apple production, which consequently affects the quality of apple fruits and causes significant economic losses. This study was aimed to investigate the influence of bitter pit on the flavonoid contents in apple fruits and its molecular biological mechanism, so as to explore the alternative bioresource of flavonoids for medical application in human chronic diseases. The total flavonoid content was determined using the aluminum chloride colorimetric method. The result showed that the total flavonoid content in bitter-pit apple fruits(BG) were 4.28-fold as that in healthy apple fruits(JKG). By RNA sequencing, 257.002 and 225.713 M raw reads containing 38.55 and 33.857 Gb raw bases were obtained from JKG and BG respectively, after trimming the adapter sequences, primers, Ns, and reads with quality scores below 30, 226.702 and 202.951 M clean reads containing32.807 G and 29.626 nucleotides were obtained respectively, their base ratios with quality values higher than 20 and 30 in reads(Q20 and Q30) were more than 98.3% and 95.0%, respectively. By miRNA sequencing, 4331 clean reads associated with 3753 target genes(2571 upregulated and 1181 downregulated) were obtained. After annotation, a total of 41894 RNAs were obtained, of which, 15852 were known transcripts and 26042 were novel transcripts. Among the obtained transcripts,there were 33582 mRNAs(12645 known and 20937 novel), 1860 lncRNAs(0 known and 1860 novel), 98 circRNAs(52 known and 46 novel), 5894 miRNAs(3017 known and 2877 novel), 322 other type of RNAs(0 known and 322 novel) and138 unknown type of RNAs(138 known and 0 novel). Based on Gene Ontology(GO) gene functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis, 2632 differentially expressed genes(DEGs) were obtained from JKG vs. BG comparison, in which, 2080 DEGs were upregulated and 552 were downregulated. Among the obtained DEGs, 26 DEGs(2 downregulated and 24 upregulated) were involved in flavonoid biosynthesis [ko00941], all of them are mRNAs, there were no lncRNAs, circRNAs and miRNAs related to flavonoid biosynthesis. Then, 8 of the 26 DEGs [CYP98 A3(1), CYP98 A3(2), BADH, DAT, MdHCT(1), MdHCT(2), CHI(1) and CHI(2)] with fold change>1.0(1.43–29.42) were selected for qRT-PCR validation, the result exhibited the consistent expression patterns with that of RNA-seq analysis(R~2=0.8551), that is, the expression of the 8 DEGs were upregulated with the BG/JKG ratio of 1.05–7.17 in JKG vs BG comparison, and the ratios of qRT-PCR relative expression level were less than the fold change of RNA-seq FPKM value except DAT. The results of this study showed that the flavonoid biosynthesis and accumulation in apple fruits was significantly stimulated by bitter pit disease, which provided new insights into the influence of bitter pit on the flavonoid biosynthesis and accumulation in apples and indicated that the bitter-pit apples could be use as the potential bioresource of flavonoids for therapeutic utilization in human chronic diseases.
引文
1 Harborne J B,Williams C A.Advances in flavonoid research since 1992.Phytochemistry,2000,55:481-504
    2 Rodriguez A,Strucko T,Stahlhut S G,et al.Metabolic engineering of yeast for fermentative production of flavonoids.Bioresour Tech,2017,245:1645-1654
    3 Liu M,Li X,Liu Y,et al.Regulation of flavanone 3-hydroxylase gene involved in the flavonoid biosynthesis pathway in response to UV-Bradiation and drought stress in the desert plant,Reaumuria soongorica.Plant Physiol Biochem,2013,73:161-167
    4 Mahunu G K,Zhang H,Apaliya M T,et al.Bamboo leaf flavonoid enhances the control effect of Pichia caribbica against Penicillium expansum growth and patulin accumulation in apples.Postharvest Biol Tech,2018,141:1-7
    5 Strick R,Strissel P L,Borgers S,et al.Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia.Proc Natl Acad Sci USA,2000,97:4780-4785
    6 Knekt P,Kumpulainen J,J?rvinen R,et al.Flavonoid intake and risk of chronic diseases.Am J Clin Nutr,2002,76:560-568
    7 Dhiman A,Nanda A,Ahmad S.A quest for staunch effects of flavonoids:Utopian protection against hepatic ailments.Arab J Chem,2016,9:S1813-S1823
    8 Raffa D,Maggio B,Raimondi M V,et al.Recent discoveries of anticancer flavonoids.Eur J Med Chem,2017,142:213-228
    9 Lin M,Zhang J,Chen X.Bioactive flavonoids in Moringa oleifera and their health-promoting properties.J Funct Foods,2018,47:469-479
    10 Wang T,Li Q,Bi K.Bioactive flavonoids in medicinal plants:Structure,activity and biological fate.Asian J Pharmac Sci,2018,13:12-23
    11 Wei M,Ma Y,Liu Y,et al.Urinary metabolomics study on the anti-inflammation effects of flavonoids obtained from Glycyrrhiza.J Chromatog B,2018,1086:1-10
    12 Bondonno N P,Bondonno C P,Ward N C,et al.The cardiovascular health benefits of apples:Whole fruit vs.isolated compounds.Trends Food Sci Tech,2017,69:243-256
    13 Liu Y,Liu S Y,Lu J F,et al.Evaluation of flavor quality and antioxidant capacity of apple fruits from three Xinjiang Red-Flesh lines(in Chinese).Sci Agric Sin,2017,50:1495-1504[刘羽,刘盛雨,卢娟芳,等.新疆红肉苹果3个品系的风味品质与抗氧化能力评价.中国农业科学,2017,50:1495-1504]
    14 Chen X S,Han M Y,Su G L,et al.Discussion on today’s world apple industry trends and the suggestions on sustainable and efficient development of apple industry in China(in Chinese).J Fruit Sci,2010,27:598-604[陈学森,韩明玉,苏桂林,等.当今世界苹果产业发展趋势及我国苹果产业优质高效发展意见.果树学报,2010,27:598-604]
    15 Chen X S,Guo W W,Xu J,et al.Genetic improvement and promotion of fruit quality of main fruit trees(in Chinese).Sci Agric Sin,2015,48:3524-3540[陈学森,郭文武,徐娟,等.主要果树果实品质遗传改良与提升实践.中国农业科学,2015,48:3524-3540]
    16 Buti M,Poles L,Caset D,et al.Identification and validation of a QTL influencing bitter pit symptoms in apple(Malus×domestica).Mol Breed,2015,35:29-39
    17 Si Y,Sankaran S.Computed tomography imaging-based bitter pit evaluation in apples.BioSyst Eng,2016,151:9-16
    18 Falchi R,D’Agostin E,Mattiello A,et al.ABA regulation of calcium-related genes and bitter pit in apple.Postharvest Biol Tech,2017,132:1-6
    19 González-Talice J,Yuri J A,del Pozo A.Relations among pigments,color and phenolic concentrations in the peel of two gala apple strains according to canopy position and light environment.Sci Horticult,2013,151:83-89
    20 Abid M,Jabbar S,Hu B,et al.Synergistic impact of sonication and high hydrostatic pressure on microbial and enzymatic inactivation of apple juice.LWT-Food Sci Tech,2014,59:70-76
    21 Xu H F,Wang N,Jiang S H,et al.Content and analysis of biosynthesis-related genes of flavonoid among four strains of Malus sieversii f.neidzwetzkyana F1 population(in Chinese).Sci Agric Sin,2016,49:3174-3187[许海峰,王楠,姜生辉,等.新疆红肉苹果杂种一代4个株系类黄酮含量及其合成相关基因表达分析.中国农业科学,2016,49:3174-3187]
    22 Fernández-Jalao I,Sánchez-Moreno C,De Ancos B.Effect of high-pressure processing on flavonoids,hydroxycinnamic acids,dihydrochalcones and antioxidant activity of apple‘Golden Delicious’from different geographical origin.Innov Food Sci Emerg Tech,2018,doi:10.1016/j.ifset.2018.06.002
    23 Musacchi S,Serra S.Apple fruit quality:Overview on pre-harvest factors.Sci Horticult,2018,234:409-430
    24 Shafiq M,Singh Z.Pre-harvest spray application of phenylpropanoids influences accumulation of anthocyanin and flavonoids in‘Cripps Pink’apple skin.Sci Horticult,2018,233:141-148
    25 Zhang M M,Wang Z H,Mao Y F,et al.Effects of different pollination combinations on the fruit quality of‘Fuji’and‘Starkrimson’apple(in Chinese).Sci Agric Sin,2018,51:3551-3560[张曼曼,王增辉,毛云飞,等.不同授粉组织对‘富士’和‘新红星’苹果品质的影响.中国农业科学,2018,51:3551-3560]
    26 DeBoever C,Reid E G,Smith E N,et al.Whole transcriptome sequencing enables discovery and analysis of viruses in archived primary central nervous system lymphomas.PLoS One,2013,8:e73956
    27 Kamala S,Makeshkumar T,Sreekumar J,et al.Whole transcriptome sequencing of diseased elephant foot yam reveals complete genome sequence of Dasheen mosaic virus.Virol Rep,2015,5:1-9
    28 Yu X M,Nie P X,Xue X M,et al.Influence of bitter pit disease on soluble solid content of apple fruits(in Chinese).Shandong Agric Sci,2016,48:105-107[余贤美,聂佩显,薛晓敏,等.苹果苦痘病对果实可溶性固形物含量的影响.山东农业科学,2016,48:105-107]
    29 Sytar O,Zivcak M,Bruckova K,et al.Shift in accumulation of flavonoids and phenolic acids in lettuce attributable to changes in ultraviolet radiation and temperature.Sci Horticult,2018,239:193-204
    30 Kong L,Zhang Y,Ye Z Q,et al.CPC:Assess the protein-coding potential of transcripts using sequence features and support vector machine.Nucleic Acids Res,2007,35:W345-W349
    31 Mortazavi A,Williams B A,McCue K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nat Methods,2008,5:621-628
    32 Xie C,Mao X,Huang J,et al.KOBAS 2.0:A web server for annotation and identification of enriched pathways and diseases.Nucleic Acids Res,2011,39:316-322
    33 Wang Y,Xu L,Chen Y,et al.Transcriptome profiling of radish(Raphanus sativus L.)root and identification of genes involved in response to lead(Pb)stress with next generation sequencing.PLoS One,2013,8:e66539
    34 Zhao Y,Yao B,Zhang M,et al.Comparative analysis of differentially expressed genes in Sika deer antler at different stages.Mol Biol Rep,2013,40:1665-1676
    35 Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,2001,25:402-408
    36 Julkunen-Tiitto R,Nenadis N,Neugart S,et al.Assessing the response of plant flavonoids to UV radiation:An overview of appropriate techniques.Phytochem Rev,2015,14:273-297
    37 Shalan H,Kato M,Cheruzel L.Keeping the spotlight on cytochrome P450.BioChim Biophys Acta(BBA)-Proteins Proteom,2018,1866:80-87
    38 Nelson D R.Cytochrome P450 diversity in the tree of life.BioChim Biophys Acta,2018,1866:141-154
    39 D’Auria J C.Acyltransferases in plants:A good time to be BAHD.Curr Opin Plant Biol,2006,9:331-340
    40 St-Pierre B,Laflamme P,Alarco A M,et al.The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer.Plant J,1998,14:703-713
    41 Sullivan M L,Zarnowski R.Red clover HCT2,a hydroxycinnamoyl-coenzyme a:malate hydroxycinnamoyl transferase,plays a crucial role in biosynthesis of phaselic acid and other hydroxycinnamoyl-malate esters in vivo.Plant Physiol,2011,155:1060-1067
    42 Ban Z,Qin H,Mitchell A J,et al.Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.Proc Natl Acad Sci USA,2018,115:E5223-E5232
    43 Joshi R K,Megha S,Rahman M H,et al.A global study of transcriptome dynamics in canola(Brassica napus L.)responsive to Sclerotinia sclerotiorum infection using RNA-Seq.Gene,2016,590:57-67
    44 Bertucci A,Pierron F,Gourves P Y,et al.Whole-transcriptome response to wastewater treatment plant and stormwater effluents in the Asian clam,Corbicula fluminea.Ecotoxicol Environ Saf,2018,165:96-106
    45 Ruan M,Liu J,Ren X,et al.Whole transcriptome sequencing analyses of DHA treated glioblastoma cells.J Neurol Sci,2019,396:247-253
    46 Zhang Y,Tan Y,Zhou X R,et al.A whole-body transcriptome analysis and expression profiling of odorant binding protein genes in Oedaleus infernalis.Comp Biochem Phys D,2018,28:134-141

中国园林博物馆北京筹备办公室 | 版权所有,未经许可严禁复制或镜像

地址:北京市丰台区射击场路15号 邮编:100072 

京ICP备19031310号     京公网安备11010602006846号