‘亚历山大’葡萄果实单萜生物合成相关基因转录及萜类物质积累规律

doi:10.3864/j.issn.0578-1752.2014.07.015

摘要/Abstract

摘要： 【目的】检测‘亚历山大’葡萄果实中萜类物质的种类和含量，分析果实发育过程中单萜类物质的积累与相关基因表达的关系，为揭示玫瑰香型葡萄香气物质的积累规律和香味育种奠定基础。【方法】从果实转色后至成熟期，每周取样一次，每次随机取样80—100粒，去籽去梗后榨汁，离心取上清液用于香气物质测定，使用顶空固相微萃取方法萃取样品中的挥发性成分，对采集到的质谱图用NIST 05谱库检索，并根据已有标样的色谱保留时间和保留指数，确定香气成分的化学组成，利用已有的化合物制备标准曲线进行定量。根据DXS的ORF序列设计引物，以充分成熟的样品RNA反转录获得的cDNA为模板，通过多次独立PCR扩增获得‘亚历山大’DXS的ORF片段，根据测序结果分析单核苷酸多态性位点。根据萜类生物合成途径中的8个关键酶基因序列设计引物，使用Ubiquitin，EF1-α和GAPDH 3个看家基因做为内参，进行实时定量PCR，检测这些基因的转录丰度。【结果】从转色后至成熟期，在检测到的所有单萜中里那醇和香叶醇的含量远高于其它单萜，多种萜类浓度逐渐上升，其中里那醇、月桂烯、柠檬烯上升6—8倍，香叶醇、萜品醇、香叶醛和萜品油烯上升2—3倍，玫瑰醚和橙花醛含量变化较小，果实中里那醇、香叶醇、氧化玫瑰和月桂烯的含量高于嗅闻阈值。在单萜生物合成早期途径中，DXS1的表达从转色开始缓慢上升，在花后15周迅速上调，上升约5倍。DXS3则从12周开始迅速上升。DXR表现出波动变化规律。HDR的表达量在前期合成酶基因中是最高的，能达到DXS1、DXS3、DXR的10—20倍；在合成途径中期的2个基因FPPS和GPPS，从转色后至成熟期表达量持续上调，FPPS的表达量上升了4倍，GPPS的表达量上升了2倍；在合成途径的后期，Liner-syn和Terp-syn在花后的11—15周都出现表达量升高，所有基因在花后17周出现明显的表达下降。单萜总量从花后12—16周迅速积累，这与同期DXS3、DXR、HDR、GPPS、FPPS的表达上调的趋势相一致。通过相关性分析结果显示萜类总量与DXS3的相关系数为0.831。‘亚历山大’DXS1的cDNA的开放阅读框全长2 151 bp，编码716个氨基酸，通过多次独立克隆后对测序结果进行比对，在该序列中发现了16个单核苷酸多态性位点，导致4个位置编码的氨基酸发生变化。【结论】单萜合成途径中多个关键酶基因后期表达上调，导致成熟过程中单萜化合物含量上升2—8倍，DXS3与单萜总量的积累具有显著的相关性。

关键词: 亚历山大葡萄 , 萜类 , 生物合成 , 反转录聚合酶链式反应

Abstract: 【Objective】 The aim of this study was to detect the terpenes and their contents in Alexandria grape, analyze the terpenes accumulation and genes transcript profiles related to their biosynthesis in developing grape berries, to elucidate the regulation of aromatic components for breeding of aromatic grapes. 【Method】 The berries were sampled once a week from veraison to full mature stage, 80-100 berries were randomly chosen, seeds and pedicels were removed, then crushed into juice, after centrifuged, the supernatant was prepared for detection of aromatic substances. Volatile components were extracted by head space solid phase micro extraction method, the compounds were identified by a combination of matching retention index and time with NIST05 library and authentic standards, the compounds were quantified using the internal standard. The primers were designed according to the ORF sequence of DXS gene, RNA of full ripening berries were reverse transcripted into cDNA, which was used as a template to amplify OFR fragments. DXS gene of Alexandria grape was obtained through several times of independent PCR, and single nucleotide polymorphism sites were found by blast of sequence results. Primers of eight key genes in the terpenes biosynthesis pathway were designed, three housekeeping genes of Ubiquitin, EF1-α and GAPDH were used as internal references, transcription abundance of these genes were detected by real-time PCR. 【Result】 The contents of linalool and geraniol were much higher than other monoterpenes, the concentration of various terpenes increased from veraison to ripening, linalool, myrcene and limonene increased by 6-8 times, geraniol, terpineol, geranial and terpinolene increased by 2 to 3 times, rose oxide and neral increased slightly. The concentrations of linalool, gerniol, rose oxide and myrcene were higher than their odor threshold. In the early pathway of monoterpenes biosynthesis, DXS1 gene was gradually up-regulated from veraison and increased by 5 times at the time of 15 weeks after blossom (15WAB). DXS3 gene was up-regulated from 12WAB and DXR gene showed a fluctuating trend, the relative expression level of HDR gene was most abundant in the early pathway and reached 10 to 20 times of DXS1, DXS3 and DXR genes. FPPS gene was up-regulated by 4 times and GPPS by 2 times in the middle pathway, Liner-syn gene and Terp-syn gene were up-regulated from 11 to 15 WAB in the late pathway, and all the genes were down-regulated significantly from 17 WAB. The total contents of monoterpenes accumulated drastically from 12 to 16 WAB, which were in accordance with the expression trend of DXS3, DXR, HDR, GPPS and FPPS genes, the correlation coefficient of total terpenes to DXS3 was 0.831. The ORF length of DXS1 gene was 2 151 bp, it encoded 716 amino acids, 16 single nucleotide polymorphism sites were found by blast of 20 independent sequence results, which caused the change of 4 amino acids.【Conclusion】In the pathway of terpenes biosynthesis, many genes were up-regulated at late-ripening stage, which led to two-eight fold increase of terpenes. DXS3 gene expression was correlated significantly to the total content of terpenes.

Key words: Alexandria grape , monotterpenes , biosynthesis , RT-PCR

孙磊1, 朱保庆2, 孙晓荣1, 许晓青3, 王晓玥1, 张国军1, 闫爱玲1, 徐海英1. ‘亚历山大’葡萄果实单萜生物合成相关基因转录及萜类物质积累规律[J]. 中国农业科学, 2014, 47(7): 1379-1386.

SUN Lei-1, ZHU Bao-Qing-2, SUN Xiao-Rong-1, XU Xiao-Qing-3, WANG Xiao-Yue-1, ZHANG Guo-Jun-1, YAN Ai-Ling-1, XU Hai-Ying-1. Terpenes Biosynthesis Related Gene Transcript Profiles and Terpenes Accumulation of ‘Alexandria’ Grape[J]. Scientia Agricultura Sinica, 2014, 47(7): 1379-1386.

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