RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响
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RNAimediated Gene Silencing of PjCAS Affects the Biosynthesis of Saponins in Panax japonicus
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    摘要:

    该研究利用Gateway技术构建珠子参环阿屯醇合成酶基因(Panax japonicus cycloartenol synthase,PjCAS)的RNAi表达载体,利用农杆菌介导转化在珠子参细胞中成功实现了PjCAS 的RNA干扰;采用实时荧光定量PCR分析珠子参皂苷生物合成途径中关键酶基因的表达情况,同时检测转基因细胞中皂苷和植物甾醇含量的变化,探讨PjCAS基因对珠子参皂苷合成的调控作用。结果表明:(1)成功获得PjCAS基因的RNAi片段,并成功构建了PjCAS基因RNAi载体pHellsgatePjCAS。(2)经农杆菌遗传转化,获得6株实现PjCAS基因RNA干扰的转基因阳性细胞系。(3)与普通细胞系相比,转基因细胞系中PjCAS基因的表达量大约下降了85%,同时与珠子参皂苷合成直接相关的关键酶基因PjDSPjAS表达量最高分别上调了90%和150%。(4)转基因细胞系中6种单体皂苷的含量均显著高于对照组,其中达玛烷型单体皂苷Re、Rb1、Rd和齐墩果烷型单体皂苷R0、IV、IVa的平均含量比普通珠子参细胞系分别提高了28%、49%、40%、36%、59%、50%。说明珠子参皂苷含量的变化受PjCAS基因的间接调控。(5)6株转基因细胞系中植物甾醇含量较对照显著降低了53%~73%。研究发现,沉默PjCAS基因可促进珠子参皂苷合成的关键酶基因PjDSPjAS显著上调表达,并提高转PjCAS基因细胞系中单体皂苷的含量,从而促进了珠子参皂苷合成量的显著增加,证明通过抑制植物甾醇合成通路关键基因PjCAS的表达可以有效降低植物甾醇合成支路的代谢通量,使更多的代谢流朝着珠子参皂苷合成方向流动,最终促进了珠子参皂苷的生物合成。

    Abstract:

    RNAi expression vector of cycloartenol synthase gene (PjCAS) from Panax japonicus was constructed by Gateway technology in this study, and Agrobacteriummediated transformation was used to realize RNAi of PjCAS in P. japonicus cells successfully. Realtime quantitative PCR was used to analyze the expression of key enzyme genes involved in the biosynthetic pathway of P. japonicus saponins (PJS), the changes of saponins and phytosterols in transgenic cells were also determined, and the regulation effect of PjCAS on the synthesis of PJS was discussed. The results showed that: (1) the RNAi fragment of PjCAS was amplified, and the PjCAS RNAi vector pHellsgatePjCAS was successfully constructed. (2) Six positive transgenic cell lines with PjCAS interferance were obtained by Agrobacterium transformation. (3) Compared with the ordinary cell line, the expression of PjCAS in transgenic cell lines was approximately decreased by 85%, and the highest expression levels of key enzyme genes PjDS and PjAS directly related to saponins synthesis were upregulated by 90% and 150%, respectively. (4) The contents of six monomeric saponin in the transgenic cell lines were significantly higher than that of control, among them, the average contents of dammaranetype monomeric saponin Re, Rb1, Rd and oleananetype monomeric saponin R0, IV, IVa were higher than that of the normal P. japonicus cells, increased by 28%, 49%, 40%, 36%, 59%, and 50%, respectively. The results indicated that the change of PJS content is indirectly regulated by PjCAS gene. (5) The phytosterol content in six transgenic cell lines decreased by 53%-73% than that in control group. Studies have found that silencing PjCAS gene can significantly upregulate the expression of key enzyme genes PjDS and PjAS related to the synthesis of PJS, and increase the content of monomeric saponin in the cell lines with PjCAS, thus promote the significant increase in the synthesis of PJS. The results prove that the metabolic flux of the branch of phytosterol synthesis could be decreased by inhibiting the expression of PjCAS, the key gene in the biosynthetic pathway of phytosterols. As a result of it, more metabolic flux flowed towards the synthesis of PJS and the biosynthsis of PJS was promoted.

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江 敏,姜 森,曲 媛,等. RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响[J].西北植物学报,2020,40(11):1816-1823

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  • 在线发布日期: 2020-12-11
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