金线莲转录组测序及其黄酮类合成相关基因分析
Analysis of transcriptome sequencing and related genes of flavonoid biosynthesis from Anoectochilus roxburghii
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摘要: 首次采用RNA-seq高通量测序技术对不同种植时期的金线莲进行转录组测序，并通过Q-PCR和HPLC对其结果进行验证和分析。转录组测序共获得金线莲51 370条基因，并注释到Nr、GO、Swiss-Prot、KEGG和KOG数据库，根据同源序列比对，与油棕和海枣的同源性最高。通过比较不同生长时间的金线莲转录组，分析其差异基因主要集中在黄酮类生物合成相关基因，运用Q-PCR对6个黄酮类生物合成相关基因(反式肉桂酸4-单加氧酶、咖啡酰辅酶A O-甲基转移酶、查尔酮合成酶、黄酮醇合成酶、莽草酸O-羟基肉桂酰转移酶、黄酮类3′，5′-羟化酶)表达量进行验证；并结合HPLC对相应的金线莲样品中6种主要黄酮类成分(芦丁、异槲皮苷、水仙苷、槲皮素、山柰酚和异鼠李素)进行含量测定。结果表明6种主要黄酮类成分含量随着金线莲黄酮类合成基因的表达量的升高而增加，并结合转录组数据，绘制出金线莲中黄酮类成分的生物合成途径。此研究为金线莲黄酮合成关键基因及黄酮类成分生物合成提供重要的遗传信息，同时为其药用价值开发提供依据。Abstract: Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii(AR)in different harvesting periods using RNA-seq high-throughput sequencing technique, and the results were verified and analyzed by Q-PCR and HPLC. A total of 51, 370 genes were obtained by transcriptome sequencing and annotated to the database of Nr, GO, Swiss-Prot, KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods, it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes(trans-cinnamate 4-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, flavonol synthase, shikimate O-hydroxycinnamoyltransferase and flavonoid 3′, 5′-hydroxylase)were verified by Q-PCR, and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids(rutin, isoquercitrin, narcissin, quercetin, kaempferol and isorhamnrtin)were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time, and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data, the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids, as well as the basis for the development of its medicinal value.
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