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  • 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
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.
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