中国农业科学 ›› 2024, Vol. 57 ›› Issue (9): 1779-1793.doi: 10.3864/j.issn.0578-1752.2024.09.012

• 园艺 • 上一篇    下一篇

基于转录组测序的苦瓜皂苷合成相关基因的鉴定和分析

戚仁洁1,2(), 宁宇3(), 刘静3, 刘之洋3, 徐海3, 罗志丹1,2(), 陈龙正3()   

  1. 1 江苏海洋大学药学院/江苏省海洋生物资源与环境重点实验室/江苏省海洋药物化合物筛选重点实验室,江苏连云港 222005
    2 江苏海洋大学/江苏海洋生物产业技术协同创新中心,江苏连云港 222005
    3 江苏省农业科学院蔬菜研究所/江苏省高效园艺作物遗传改良重点实验室,南京 210014
  • 收稿日期:2023-09-29 接受日期:2023-12-18 出版日期:2024-05-01 发布日期:2024-05-09
  • 通信作者:
    罗志丹,E-mail:
    陈龙正,E-mail:
  • 联系方式: 戚仁洁,E-mail:13956705571@163.com。宁宇,E-mail:20190022@jaas.ac.cn。戚仁洁和宁宇为同等贡献作者。
  • 基金资助:
    国家自然科学基金(32102392); 江苏省重点研发计划(Be2022339); 江苏省自然科学基金(BK20200279); 江苏省自然科学基金(3027)

Identification and Analysis of Genes Related to Bitter Gourd Saponin Synthesis Based on Transcriptome Sequencing

QI RenJie1,2(), NING Yu3(), LIU Jing3, LIU ZhiYang3, XU Hai3, LUO ZhiDan1,2(), CHEN LongZheng3()   

  1. 1 College of Pharmacy/Jiangsu Key Laboratory of Marine Biological Resources and Environment/Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, Jiangsu
    2 Co-Innovation Center of Jiangsu Marine Bio-industry Technology/Jiangsu Ocean University, Lianyungang 222005, Jiangsu
    3 Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences /Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014
  • Received:2023-09-29 Accepted:2023-12-18 Published:2024-05-01 Online:2024-05-09

摘要:

【目的】皂苷是苦瓜果实苦味的主要来源,具有降血糖、抗癌等多种药用价值。鉴定和挖掘参与调控苦瓜皂苷生物合成的代谢通路及基因,为进一步深入解析苦瓜皂苷形成的分子机制提供参考。【方法】以苦瓜高代自交系GK24为试验材料,分别在子房期(T1)、幼果期(T2)、商品果期(T3)和完全成熟期(T4)取果实组织,通过香草醛-冰醋酸法测定不同时期的苦瓜皂苷含量,利用转录组测序的方法鉴定差异表达基因。【结果】从T1到T4,苦瓜内源皂苷含量随着果实发育而呈现显著下降的趋势。转录组测序共鉴定到17 504个基因,在T1-vs-T2、T2-vs-T3和T3-vs-T4三组比较中,下调表达基因数量均高于上调表达基因数量。GO富集分析表明含磷复合物代谢过程、驱动蛋白复合体和2-琥珀酰-6-羟基-环己二烯-1-羧酸合成酶活性分别是生物过程、细胞组分及分子功能模块最显著富集的条目。KEGG分析显示全局与概述图谱、碳水化合物代谢和氨基酸代谢是3组比较中共同的最显著富集的代谢通路。根据表达模式可将基因划分为20个模块,其中profile 0中基因的表达量从T1到T4逐渐降低,与苦瓜果实发育过程中皂苷含量变化规律一致。从profile 0中鉴定出与苦瓜皂苷生物合成相关的基因14个,包括萜类骨架生物合成通路上的基因AAT1HMG1MVKPMKMVD2FPS1,倍半萜与三萜生物合成途径中的基因SS12SQE1CPQ及后修饰酶基因CYP97A3CYP71AN24UGT94E5UGT73C6。实时荧光定量PCR(qRT-PCR)结果与RNA-seq结果一致。【结论】苦瓜果实中皂苷含量随着果实不断成熟而逐渐降低。苦瓜皂苷生物合成首先通过上游的萜类骨架生物合成(ko00900)代谢途径完成萜类皂苷骨架的积累,随后通过倍半萜与三萜生物合成(ko00909)代谢途径以及下游的氧化还原酶、糖基转移酶的修饰最终生成皂苷,本研究通过转录组测序共鉴定到14个与苦瓜皂苷生物合成相关的基因。

关键词: 苦瓜, 转录组, 差异表达基因, 皂苷, 合成调控

Abstract:

【Objective】As the main source of bitterness in bitter gourd fruit, saponin possesses various medicinal values including hypoglycemic and anticancer properties. This paper aimed at identifying the metabolic pathways and genes involved in the regulation of bitter gourd saponin biosynthesis, so as to provide the theoretical basis for further analysis of the molecular mechanism of bitter gourd saponin formation.【Method】Using the bitter gourd high-generation inbred line GK24 as the test material, fruit tissues were sampled at the ovary stage (T1), young fruit stage (T2), commodity fruit stage (T3), and maturity fruit stage (T4). The saponin content of bitter gourd at different periods were determined by the vanillin-glacial acetic acid method, and the differentially expressed genes (DEGs) were identified using the method of transcriptome sequencing. 【Result】From T1 to T4, the endogenous saponin content of bitter gourd showed significant decrease with fruit development. A total of 17 504 genes were identified by transcriptome sequencing, and the number of down-regulated genes was higher than that of up-regulated genes in the comparison of the three groups of T1-vs-T2, T2-vs-T3, and T3-vs-T4. GO enrichment analysis showed that phosphorus-containing complex metabolic process, kinesin complex and 2-succinyl-6-hydroxy-cyclohexadiene-1-carboxylic acid synthetase activity were the most significantly enriched terms in biological process, cellular component and molecular function, respectively. KEGG analysis showed that global and overview mapping, carbohydrate metabolism, and amino acid metabolism were the most significantly enriched pathways in all three comparative groups. The genes could be categorized into 20 profiles based on their expression patterns. Genes in profile 0 gradually decreased from T1 to T4, showing similar pattern of saponin content changes with bitter gourd fruit development. 14 genes related to bitter gourd saponin biosynthesis were identified from profile 0, including AAT1, HMG1, MVK, PMK, MVD2, and FPS1 in the terpene skeleton biosynthesis pathway, SS12, SQE1, and CPQ in the sesquiterpene and triterpene biosynthesis pathway, and the postmodifying enzyme genes CYP97A3, CYP71AN24, UGT94E5 and UGT73C6. The results of RNA-seq were validated by qRT-PCR method.【Conclusion】The saponin content of bitter gourd decreased with the fruit development. Bitter gourd saponin was synthesized through the terpenoid bone biosynthesis (ko00900) metabolic pathway to accumulate terpenoid saponin skeleton in the first, and then produce saponins through the sesquiterpene and triterpene biosynthesis (ko00909) metabolic pathway and the modification of oxidoreductase and glycosyltransferase successively. A total of 14 genes related to the saponin biosynthesis of bitter gourd have been identified in the present study.

Key words: bitter gourd, transcriptome, differentially expressed genes, saponin, regulation of synthesis