褪黑素通过诱导STS基因表达提高了葡萄白藜芦醇含量

摘要/Abstract

摘要： 摘要：【目的】查明外源褪黑素处理是否能够诱导葡萄茋类合酶基因（Stilbene synthase, STS）表达，进而提高葡萄白藜芦醇含量；为应用褪黑素来提高葡萄营养价值和揭示褪黑素调控白藜芦醇代谢的分子机理提供理论基础。【方法】利用100 μmol·L-1的褪黑素加表面活性剂浸泡转色早期的葡萄果穗，利用30 μmol·L-1的褪黑素处理生长30d左右的无菌组培苗根系。利用葡萄基因组数据库，结合RNA-Seq技术，鉴定葡萄上褪黑素诱导的STS基因家族成员；并利用Clustalx和MEGA5软件进行序列分析和构建进化树，利用在线软件plantCARE分析STS基因启动子区域的顺式作用元件。利用实时荧光定量RT-PCR检测STS基因在褪黑素处理不同时间点的表达水平；利用高效液相色谱测定白藜芦醇含量。【结果】RNA-Seq分析表明葡萄果实中有30个STS基因被褪黑素处理诱导上调，占葡萄总STS基因的62.5%，占总上调基因的8.5%。褪黑素诱导的STS基因主要定位在16号染色体上，氨基酸序列高度保守，相似度达88.3%，同源性最高的STS蛋白间仅有1个氨基酸差异。启动子序列分析表明STS基因具有相对保守的启动子序列，相似度在55.6%以上。根据蛋白质和启动子序列相似度，30个STS基因均可划分为A、B、C三组，并且二者聚类结果基本一致。在A和B组STS启动子区域分别发现了一段高度保守的序列。作用元件分析表明，在STS启动子区存在ABA、乙烯、水杨酸和生长素等激素响应元件和MYB结合位点。褪黑素诱导的30个STS基因在不同组织中具有不同的表达水平，总体上在根系中表达水平较高；果实中不同STS基因具有不同的转录丰度。通过定量PCR检测了5个STS基因在褪黑素处理不同时间的表达水平与RNA-Seq结果一致，均受褪黑素诱导，但具有不同的表达响应模式。【结论】褪黑素诱导上调了30个葡萄STS基因，它们之间具有保守的蛋白序列和启动子序列。30个STS基因启动子区域含有不同的作用元件，对褪黑素具有不同的表达响应模式。褪黑素处理能提高葡萄果实和根系中白藜芦醇含量。

关键词: 关键词：葡萄, STSs, 序列特征, 表达分析, 白藜芦醇

Abstract: Abstract：【Objective】 This study was aimed to investigate whether melatonin treatment could induce the expression of STSs and thereby enhance content of resveratrol in grapevine. This study will lay a basis for the application of melatonin in improving grape berry nutrition value and shed light on the molecular mechanism underlying the regulation of melatonin on resveratrol metabolism 【Method】The whole grape clusters were treated with 100 μmol·L-1 melatonin at early veraison, and the roots of in vitro shoot cultures were treated with 30 μmol·L-1 melatonin at approximately 30 days after subculture. RNA-Seq with the grape genome data as a reference was employed to identify melatonin-induced STS genes. Sequence and clustering analysis were performed using Clustalx and MEGA5 softwares, and the cis-acting elements in the promoter region were identified using online plantCARE software. The expression levels of STS genes were detected using quantitative real-time RT-PCRs. The content of resveratrol was determined by high performance liquid mass spectrometry (HPLC). 【Result】RNA-Seq analysis showed that 30 STSs were up-regulated by melatonin treatment, accounting for 62.5% of the total amount of grape STS genes and 8.5% of all the up-regulated genes. The melatonin-induced STSs primarily located in chr 16 and their deduced proteins possess more than 88.3% similarity; only one different amino acid existed between the two STSs with the highest similarity. The promoter regions of different STSs were also highly conserved and the similarity surpassed 55.6%. The detected 30 STSs were clustered into three groups, i.e., A, B and C, in the light of their sequence similarity of protein or promoter. A highly conserved sequence was found in the promoter of STSs from groups A and B. Additionally, there were a lot of cis-acting elements related to ABA, ethylene, salicylic acid and auxin in the promoters of STSs. The 30 melatonin-induced STSs exhibited varying expression patterns in different tissues and generally they were predominantly expressed in grape roots; additionally, they have varying transcriptional abundance in grape berries. qRT-PCRs showed that the 5 selected STSs were induced by melatonin, consistent with the results of RNA-Seq, and possessed different expression patterns in response to melatonin.【Conclusion】A total of 30 STSs were induced by melatonin in grape berries and they possessed highly conserved protein and promoter sequences. The promoter regions of 30 STSs contained different cis-acting elements and they exhibited different expression patterns in response to melatonin treatment. Melatonin treatment enhanced the content of resveratrol in grape berries and roots.

Key words: Key words: grape, STS, sequence characteristics, expression analysis, resveratrol

中图分类号:

S663.1

许丽丽姚玉新. 褪黑素通过诱导STS基因表达提高了葡萄白藜芦醇含量[J]. 食品科学, 0, (): 0-0.

xin yuyao. Melatonin treatment enhances content of resveratrol via up-regulating STS expression in grapevine[J]. FOOD SCIENCE, 0, (): 0-0.

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