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首页> 外文期刊>BMC Genomics >An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis
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An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

机译：RNA-seq和数字基因表达分析发现罗汉果罗汉松三萜生物合成基因的有效方法

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Background Siraitia grosvenorii (Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in S. grosvenorii, especially the late steps of the pathway. Results In this study, a cDNA library generated from of equal amount of RNA taken from S. grosvenorii fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven CYP450s and five UDPGs were selected as the candidates most likely to be involved in mogrosides biosynthesis. Conclusion A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven CYP450s and five UDPGs were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from S. grosvenorii.
机译：背景罗汉果（Siraitia grosvenorii，罗汉果）是多年生草本植物，原产于中国南方，在桂林市最为流行。其果实含有甜，肉质，可食用的果肉，被广泛用于传统中药。水果提取物中的主要生物活性成分是葫芦巴型三萜皂苷，被称为罗汉果苷。其中，罗汉果甙V的甜度是蔗糖的近300倍。然而，关于罗汉果罗汉果中的罗汉果苷生物合成知之甚少，特别是该途径的后期。结果在本研究中，使用Illumina / Solexa平台对从开花后50天（DAF）和70 DAF提取的罗汉果果实等量RNA生成的cDNA文库进行了测序。从cDNA文库产生了超过48,755,516个高质量读段，这些读段被组装成43,891个单基因。从头组装和缺口填充产生了43,891个单基因，平均序列长度为668个碱基对。京都基因与基因组百科全书对总共26,308（59.9％）个独特序列进行了注释，并将其中11,476个独特序列分配给了特定的代谢途径。从我们的文库中鉴定出了与罗汉果苷骨架合成有关的所有已知酶的cDNA序列。此外，总共鉴定出八十五种细胞色素P450（CYP450）和九十个UDP-葡萄糖基转移酶（UDPG）单基因，其中一些似乎编码负责将罗汉果苷骨架转化为各种罗汉果苷的酶。使用Solexa测序对水果发育的三个重要阶段进行了数字基因表达谱（DGE）分析，根据它们的表达模式，选择了7种CYP450和5种UDPG作为最可能参与罗汉果苷生物合成的候选对象。结论基于下一代测序技术的RNA-seq和DGE分析相结合，是一种鉴定候选基因的有效方法，该候选基因编码负责非模式植物中新型次生代谢产物生物合成的酶。选择了7种CYP450和5种UDPG作为罗汉果苷生物合成的潜在候选物。这项研究的转录组数据为了解罗汉果果实提取物中主要生物活性成分的形成提供了重要资源。

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