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作物杂志,2024, 第1期: 48–56 doi: 10.16035/j.issn.1001-7283.2024.01.007

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

过氧化氢诱导膜荚黄芪不定根毛蕊异黄酮葡萄糖苷积累的转录组分析

窦维泽1(), 姜雯2, 冯艺川2, 金卓2, 全雪丽2, 吴松权1,2()   

  1. 1延边大学融合学院，133000，吉林延吉
    2延边大学农学院，133000，吉林延吉
  • 收稿日期:2023-05-31 修回日期:2023-09-01 出版日期:2024-02-15 发布日期:2024-02-20
  • 通讯作者: 吴松权，研究方向为特种植物资源与利用，E-mail：arswsq@ybu.edu.cn
  • 作者简介:窦维泽，研究方向为特种植物资源与利用，E-mail：1424327165@qq.com
  • 基金资助:
    吉林省自然科学基金(YDZJ202101ZYTS197);国家自然科学基金(21462044)

Transcriptome Analysis Reveals Mechanisms of Calycosin- 7-O-β-D-Glucoside Accumulation in Astragalus membranaceus Adventitious Roots through Hydrogen Peroxide

Dou Weize1(), Jiang Wen2, Feng Yichuan2, Jin Zhuo2, Quan Xueli2, Wu Songquan1,2()   

  1. 1College of Integration Science, Yanbian University, Yanji 133000, Jilin, China
    2Agricultural College, Yanbian University, Yanji 133000, Jilin, China
  • Received:2023-05-31 Revised:2023-09-01 Online:2024-02-15 Published:2024-02-20
  • Contact: Wu Songquan

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摘要：

H2O2处理能显著增加膜荚黄芪不定根中毛蕊异黄酮葡萄糖苷（CG）的含量。为探究其生物合成机制，以膜荚黄芪不定根为试验材料，经H2O2处理后进行转录组测序筛选差异表达基因。选取上调基因进行功能和代谢通路分析，用qRT-PCR验证，并对相关生理生化指标进行测定。结果表明，差异表达基因的筛选和基因功能注释表明，H2O2处理引起了氧化胁迫，为抵消氧化胁迫，膜荚黄芪不定根通过激活乙烯信号通路，诱导苯丙烷异黄酮生物合成途径中的全部结构基因，从而显著增加CG的含量。此外，为了增加CG，H2O2处理也会诱导苯丙氨酸的生物合成。试验结果进一步揭示了膜荚黄芪不定根中CG的积累机制，为今后规模化生产黄芪CG奠定理论依据。

关键词: H2O2, 氧化胁迫, 膜荚黄芪, 转录组测序, 毛蕊异黄酮葡萄糖苷

Abstract:

Hydrogen peroxide (H2O2) significantly enhanced the content of calycosin-7-O-β-D-glucoside (CG) in Astragalus membranaceus adventitious roots (AMARs). To discover its biosynthetic mechanism of CG, AMARs were used to screen the differentially expressed genes (DEGs) through transcriptome sequencing after H2O2 treatment. The function and metabolic pathways were analyzed by using up-regulated genes, verified them by qRT-PCR method, and measured the relevant physiological and biochemical parameters. The results showed that AMARs activated ethylene signaling, induced all structural genes in the phenylpropane-isoflavone biosynthesis pathway, significantly increased the content of CG to counteract the oxidative stress caused by H2O2 treatment. Furthermore, H2O2 treatment also induced phenylalanine biosynthesis in order to increase the content of CG. These results revealed the accumulation mechanism of CG in AMARs which laid a theoretical basis for large-scale production of CG in the future.

Key words: H2O2, Oxidative stress, Astragalus membranaceus (Fisch.) Bunge, Transcriptome sequencing, Calycosin-7-O-β-D-glucoside

表1

测序数据质量评估统计

样品
Sample		 Reads总数
Sum of
reads		 碱基总数
Sum of
base		 GC含量
GC
content
(%)		 碱基质量值
Quality score
of bases (%)
Q20 Q30
A1 21 235 913 6 331 453 200 42.92 98.00 94.15
A2 21 342 976 6 357 785 560 42.69 97.96 94.03
A3 22 438 643 6 700 654 314 42.65 97.80 93.64
B1 23 860 654 7 110 359 156 42.73 97.93 93.93
B2 21 448 660 6 403 522 654 42.69 97.71 93.38
B3 21 112 504 6 297 173 426 42.84 97.67 93.40
C1 21 096 613 6 286 682 948 42.62 97.66 93.33
C2 22 710 737 6 770 120 316 42.61 97.73 93.51
C3 21 934 340 6 540 593 324 42.74 98.05 94.20
D1 21 991 682 6 548 307 308 42.63 97.95 94.00
D2 23 063 182 6 885 014 356 42.80 97.84 93.77
D3 19 383 999 5 780 350 228 42.76 97.79 93.64

表2

H2O2处理下差异表达基因数量

分组
Group		 差异基因总数
Total number
of different
genes		 上调基因数
Number of
up-regulated
genes		 下调基因数
Number of
down-regulated
genes
B vs A 222 206 16
C vs A 468 347 121
D vs A 280 199 81
A vs B & C & D 135 127 8

图1

差异表达基因的GO功能分类图

图2

差异表达基因的KEGG富集图 (a) H2O2处理1 h，(b) H2O2处理3 h，(c) H2O2处理6 h。

图3

异黄酮分支途径合成基因的相对表达量 不同字母表示不同处理时间下的显著性差异（P < 0.05），“*”表示对照和H2O2处理之间的显著性（P < 0.05），下同。

图4

异黄酮分支途径差异表达基因聚类热图不同颜色区域分别代表不同的聚类分组信息，颜色由红到蓝表示差异表达基因的表达量由高到低。

图5

H2O2处理对CG含量的影响

图6

POD的相对表达量及活性

图7

ACS和ERF的相对表达量及H2O2处理对乙烯含量的影响

图8

苯丙氨酸合成途径中基因的相对表达量

[1] 顾志荣, 葛斌, 许爱霞, 等. 基于本草考证的黄芪功效主治及用药禁忌挖掘. 中成药, 2018, 40(11):2524-2530.
[2] 王文兰, 陶波. 黄芪炮制方法历史沿革及研究进展. 亚太传统医药, 2015, 11(6):38-40.
[3] 刘丽, 林庶如, 李军, 等. 毛蕊异黄酮葡萄糖苷与葛根素及其配伍对3T3-L1前脂肪细胞胰岛素抵抗细胞模型的影响. 中药药理与临床, 2018, 34(1):10-14.
[4] Zhang L, Gong A, Riza K, et al. A novel combination of four flavonoids derived from Astragali Radix relieves the symptoms of cyclophosphamide-induced anemic rats. FEBS Open Bio, 2017, 7(3):303-433.
doi: 10.1002/feb4.2017.7.issue-3
[5] Liu M H, Li P L, Zeng X, et al. Identification and pharmacokinetics of multiple potential bioactive constituents after oral administration of Radix Astragali on cyclophosphamide- induced immunosuppression in Balb/cmice. International Journal of Molecular Sciences, 2015, 16(3):5047-5071.
doi: 10.3390/ijms16035047
[6] 张兰涛, 郭宝林, 朱顺昌, 等. 黄芪种质资源调查报告. 中药材, 2006, 29(8):771-773.
[7] 秦嘉泽, 全雪丽, 田海丽, 等. 长白山野生膜荚黄芪不定根总黄酮积累规律研究. 北方园艺, 2014(16):158-160.
[8] Jin H Y, Yu Y, Quan X L, et al. Promising strategy for improving calycosin-7-O-β-D-glucoside production in Astragalus membranaceus adventitious root cultures. Industrial Crops and Products, 2019, 141:111792.
doi: 10.1016/j.indcrop.2019.111792
[9] Trapnell C, Williams B A, Pertea G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 2010, 28(5):511-515.
doi: 10.1038/nbt.1621 pmid: 20436464
[10] 郑涵予. 氮、磷及糖浓度对膜荚黄芪不定根毛蕊异黄酮葡萄糖苷积累的影响. 延吉:延边大学, 2020.
[11] 张恒.星星草(Puccinellia tenuiflora)叶绿体Na2CO3胁迫应答的生理学与定量蛋白质组学研究. 哈尔滨:东北林业大学, 2012.
[12] 刘丰娇. 氧化还原信号参与硫化氢对低温下黄瓜光合作用的调控. 泰安:山东农业大学, 2020.
[13] Chen J, Zhang H P, Feng M F, et al. Transcriptome analysis of woodland strawberry (Fragaria vesca) response to the infection by Strawberry vein banding virus (SVBV). Virology Journal, 2016, 13(1):128-134.
doi: 10.1186/s12985-016-0584-5
[14] Shi Y T, Tian S W, Hou L Y, et al. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. The Plant Cell, 2012, 24(6):2578-2595.
doi: 10.1105/tpc.112.098640 pmid: 22706288
[15] 李建, 黄琳丽. 乙烯在植物抗性反应中的作用. 生物化工, 2020, 6(6):140-142.
[16] 高佳钰, 游晓商, 张瞳, 等. 黄梁木ACS基因家族鉴定及其表达模式分析. 分子植物育种, 2023, 21(7):2200-2210.
[17] Wang Z Y, Yadav V, Yan X, et al. Systematic genome-wide analysis of the ethylene-responsive ACS gene family: contributions to sex form differentiation and development in melon and watermelon. Gene, 2021, 805:145910.
doi: 10.1016/j.gene.2021.145910
[18] 刘同金, 徐铭婕, 崔群香, 等. 萝卜乙烯合成途径基因的鉴定及对胁迫的响应. 西北植物学报, 2022, 42(4):558-568.
[19] 宦晨. 桃果实发育和贮藏期间抗氧化系统对活性氧水平的调控机理. 南京:南京农业大学, 2018.
[20] 王坤. 柿果实乙烯合成启动与氧化还原电位及相关酶活性的关系. 南宁:广西大学, 2017.
[21] Liu M C, Pirrello J, Chervin C, et al. Ethylene control of fruit ripening: revisiting the complex network of transcriptional regulation. Plant Physiology, 2015, 169(4):2380-2390.
doi: 10.1104/pp.15.01361 pmid: 26511917
[22] Feng K, Hou X L, Xing J X, et al. Advances in AP2/ERF super-family transcription factors in plant. Critical Reviews in Biotechnology, 2020, 40(6):750-776.
doi: 10.1080/07388551.2020.1768509 pmid: 32522044
[23] Rehman S, Mahmood T. Functional role of DREB and ERF transcription factors: regulating stress-responsive network in plants. Acta Physiologiae Plantarum, 2015, 37(9):178-189.
doi: 10.1007/s11738-015-1929-1
[24] 徐丽艳, 纪明山. 植物次生代谢物的类型及其对植物自身的作用. 中国农村小康科技, 2006(9):52-54.
[25] Ferreres F, Figueiredo R, Bettencourt S, et al. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2affair?. Journal of Experimental Botany, 2011, 62(8):2841-2854.
doi: 10.1093/jxb/erq458
[26] Nakabayashi R, Yonekura-Sakakibara K, Urano K, et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. The Plant Journal: for Cell and Molecular Biology, 2014, 77(3):367-379.
doi: 10.1111/tpj.2014.77.issue-3
[27] Dastmalchi M, Chapman P, Yu J, et al. Transcriptomic evidence for the control of soybean root isoflavonoid content by regulation of overlapping phenylpropanoid pathways. BMC Genomics, 2017, 18(1):70-84.
doi: 10.1186/s12864-016-3463-y pmid: 28077078
[28] 李亮亮, 黄金智. 毛蕊异黄酮葡萄糖苷药理作用的研究进展. 海南医学院学报, 2020, 26(2):156-160.
[29] 李文倩. 黄芪根转录组学研究及类黄酮合成关键基因的筛选与表达. 太原:山西农业大学, 2020.
[30] 张莹. 黄芪转录组数据分析及皂苷合成途径关键基因的表达. 太原:山西农业大学, 2020.
[31] Dong N Q, Lin H X. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions. Journal of Integrative Plant Biology, 2021, 63(1):180-209.
doi: 10.1111/jipb.v63.1
[32] 陈克杰. 大肠杆菌苯丙氨酸合成途径中关键基因的组合表达研究. 广州:华南理工大学, 2016.
[33] 刘瑞霞. E.coli中D-苯丙氨酸代谢合成的研究. 无锡:江南大学, 2015.
[34] 李晓萍, 边英男, 郝瑞昕, 等. 利用DNA shuffling构建部分解除对氟苯丙氨酸反馈抑制的aroG. 复旦学报(自然科学版), 2010, 49(5):568-574.
[35] 孙盼星, 李锋, 宗奕吾, 等. 微生物细胞工厂合成甲基萘醌的研究进展. 科学通报, 2022, 67(34):4055-4067.
[36] 张春花, 赵智, 张英姿, 等. 北京棒杆菌DAHP合成酶Ⅰ基因的克隆、序列分析及表达. 微生物学报, 2008, 48(11):1466-1472.
[37] 邢懿. 烟草谷草转氨酶基因的克隆与酶学分析. 合肥:安徽农业大学, 2017.
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