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Archive > Volume 11 Issue 2 > 2019,11(2):192-199. DOI:10.1016/j.chmed.2019.03.001 Prev Next

根特異性過(guò)表達(dá)β-香樹(shù)脂醇合成酶基因增加烏拉爾甘草毛狀根中甘草酸的含量

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Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of Glycyrrhiza uralensis

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    摘要:
    目的：烏拉爾甘草（Glycyrrhizia uralensis）是中國(guó)最常用的中藥材之一，主要在中國(guó)種植。然而，栽培的烏拉爾甘草中甘草酸含量遠(yuǎn)低于中國(guó)藥典規(guī)定的最低標(biāo)準(zhǔn)。在本文中，我們采用過(guò)表達(dá)β-香樹(shù)脂醇合成酶基因（GuBAS）的方法來(lái)增加甘草酸含量。 方法：構(gòu)建根特異性過(guò)表達(dá)GuBAS基因的植物表達(dá)載體，并以電轉(zhuǎn)法轉(zhuǎn)化到發(fā)根農(nóng)桿菌中構(gòu)建發(fā)根農(nóng)桿菌工程菌。重組發(fā)根農(nóng)桿菌工程菌侵染烏拉爾甘草的胚軸和子葉以誘導(dǎo)毛狀根。誘導(dǎo)獲得的毛狀根經(jīng)過(guò)培養(yǎng)后，采用HPLC法測(cè)定甘草酸含量。 結(jié)果：PCR和測(cè)序結(jié)果均顯示獲得了3個(gè)轉(zhuǎn)基因毛狀根系。通過(guò)qRT-PCR測(cè)定不同轉(zhuǎn)基因毛狀根系中的GuBAS拷貝數(shù)設(shè)為3，7和4。通過(guò)HPLC檢測(cè)甘草酸含量，結(jié)果顯示甘草酸僅存在于三個(gè)轉(zhuǎn)基因毛狀根系中，野生型毛狀根中未測(cè)得。 結(jié)論：根特異性過(guò)表達(dá)β-香樹(shù)脂醇合成酶基因增加了烏拉爾甘草毛狀根中甘草酸的含量。

    Abstract:
    Objective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene (GuBAS). Methods: Tobacco root-specific promoter TobRB7 and GuBAS cDNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC. Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of GuBAS in these transgenic hairy roots was intended by qRT-PCR to be 3, 7, and 4. GA was detected in the three transgenic hairy roots but in wild hairy roots. Conclusion: Over-expressing GuBAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

    關(guān)鍵詞:
    β-香樹(shù)脂醇合成酶; 烏拉爾甘草; 毛狀根; 根特異性; 過(guò)表達(dá)

    Keywords:


    Project Supported:
    National Nature Science Foundation of China (81503181)

    Clc Number:

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