中国中药杂志

2020, v.45(14) 3422-3431

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新疆紫草中2条CYP450基因的干扰毛状根体系的建立及其影响研究
Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence

梁玖雯;李锬;王瑞杉;周利;杨全;王升;郭兰萍;
LIANG Jiu-wen;LI Tan;WANG Rui-shan;ZHOU Li;YANG Quan;WANG Sheng;GUO Lan-ping;Guangdong Provincial Research Center on Good Agricultural Practice & Comprehensive Agricultural Development Engineering Technology of Cantonese Medicinal Materials, College of Traditional Chinese Medicine, Guangdong Pharmaceutical University;State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;

摘要(Abstract):

该研究依托新疆紫草悬浮细胞转录组数据库,以CYP76B74蛋白序列为搜索序列,挖掘了2条与紫草素生物合成下游途径相关的CYP450候选基因。构建了候选基因的干扰型毛状根并进行培养,测定了鲜重、干重、总萘醌含量、紫草素类化合物含量以及紫草素生物合成途径关键酶基因的表达量,初步探究了候选基因对新疆紫草毛状根的生长和紫草素合成产生的影响,并讨论了其影响紫草素合成的可能调控机制。通过本地Blast和系统发育分析,筛选出2条新疆紫草中与CYP76B74具有高同源性的CYP450候选基因(CYP76B75,CYP76B100),并构建了相应的干扰型毛状根。相比空载毛状根(RNAi-control),CYP76B75被干扰毛状根(RNAi-CYP76B75)和CYP76B100被干扰毛状根(RNAi-CYP76B100)的鲜重显著减少,而干重未受到影响,折干率显著增加。除β-乙酰氧基异戊酰阿卡宁在3组毛状根中含量都很高外,紫草素、去氧紫草素、乙酰紫草素、β,β′-二甲基丙烯酰阿卡宁、β-羟基异戊酰紫草素、异丁酰紫草素以及总萘醌在3组毛状根中的含量高低均呈现一致规律:RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control。其中,干扰CYP76B75表达后对β-羟基异戊酰紫草素合成的促进作用最显著,RNAi-CYP76B75中β-羟基异戊酰紫草素的含量为RNAi-control的11.7倍。实时荧光定量PCR结果显示,与RNAi-control相比较,RNAi-CYP76B75与RNAi-CYP76B100中AePGT基因的表达量无明显变化,CYP76B74与AeHMGR的表达量上调;此外,RNAi-CYP76B75中,CYP76B100的表达量下调,而RNAi-CYP76B100中,CYP76B75的表达量反而显著上调。结果说明CYP76B75与CYP76B100被干扰表达后,毛状根的生长受到了抑制,但却显著促进了其紫草素的合成,其可能通过上调新疆紫草毛状根中CYP76B74基因的表达量调控了紫草素生物合成。
In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β′-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. Results of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

关键词(KeyWords): 新疆紫草;紫草素;细胞色素P450;转录组;RNA干扰;毛状根
Arnebia euchroma;shikonin;cytochrome P450;transcriptome;RNA interference;hairy root

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81703648,81603239,81891014);; 中央级公益性科研院所基本科研业务费专项(ZZXT201901,ZZ13-YQ-084,ZZ13-YQ-092);; 国家重点研发计划项目(2017YFC1700701,2017YFC1701405);; 现代农业产业技术体系建设专项(CARS-21)

作者(Author): 梁玖雯;李锬;王瑞杉;周利;杨全;王升;郭兰萍;
LIANG Jiu-wen;LI Tan;WANG Rui-shan;ZHOU Li;YANG Quan;WANG Sheng;GUO Lan-ping;Guangdong Provincial Research Center on Good Agricultural Practice & Comprehensive Agricultural Development Engineering Technology of Cantonese Medicinal Materials, College of Traditional Chinese Medicine, Guangdong Pharmaceutical University;State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;

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