金鱼草AmPDS基因克隆及调节类胡萝卜素合成功能分析
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北京市教委自然科学基金委联合科技计划重点项目(KZ201800200)


Cloning of AmPDS Gene in Snapdragon and Analysis of Its Function to Regulate Carotenoid Synthesis
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    摘要:

    八氢番茄红素脱氢酶(phytoene desaturase,PDS)是类胡萝卜素进行生物合成途径中的关键酶。为了深入探究金鱼草PDS基因的功能,该研究以‘马里兰’金鱼草(Antirrhinum majus ‘Maryland True Pink’)为材料,对其PDS基因(AmPDS)全长序列及蛋白结构进行分析,并克隆了AmPDS基因片段;采用qRTPCR技术检测AmPDS基因在不同时期及部位的相对表达水平,利用VIGS技术验证AmPDS基因功能,用紫外分光法测定叶片中各类色素含量。结果显示:(1) 成功克隆AmPDS基因片段(500 bp); AmPDS基因cDNA全长1 743 bp,编码580个氨基酸;其蛋白分子量为64.75 kD,理论等电点6.66;同源比对分析显示AmPDS基因与芝麻(Sesamum indicum)的序列相似性最高。(2) qRTPCR分析表明,AmPDS基因在全株均有表达,且在全盛期花朵的上瓣和叶片中表达量最高。(3)构建pTRV2AmPDS载体,建立了金鱼草的VIGS沉默体系,AmPDS基因沉默效率约为53%,与阴性对照相比叶片中各类色素含量均显著降低。研究认为,AmPDS基因是金鱼草类胡萝卜素生物合成途径中的关键基因,可作为金鱼草VIGS沉默体系的指示基因,为后续研究金鱼草其他基因功能奠定基础。

    Abstract:

    Phytoene desaturase (PDS) is the key enzyme in the biosynthesis pathway of carotenoids. In order to further explore the functions of PDS gene in Antirrhinum majus, the fulllength sequence and protein structure of AmPDS were analyzed, and the gene fragment of AmPDS was cloned. To investigate the expression profiles of AmPDS genes, the relative expression level of AmPDS gene in different periods and organs was detected by qRTPCR, the function of AmPDS gene was verified by VIGS, and the content of various pigments in leaves was determined by UV spectrophotometry. The results showed that: (1) the AmPDS gene forgment (500 bp) was cloned correctly, the cDNA of AmPDS gene was 1 743 bp in length, encoding 580 amino acids; Its protein molecular weight was 64.75 kD, and the the theoretical isoelectric point was 6.66. Homology analysis showed that the AmPDS gene has the highest sequence similarity to the sesame (Sesamum indicum). (2) qRTPCR analysis showed that the AmPDS gene was expressed in the whole plant, and the highest expression level was found in the upper lobes of the flower in fully opened period and leaves. (3) The pTRV2AmPDS vector was successfully constructed and the VIGS system of snapdragon was established. The silencing efficiency of AmPDS gene was about 53% while the contents of various pigments in the leaves were significantly reduced comparing with the negative control. AmPDS is a key gene in the carotenoid biosynthesis pathway of snapdragon, and its obvious phenomenon can be used as an indicator gene for the VIGS system of snapdragon, which can lay a foundation for the further study of other gene functions of snapdragon.

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韩嘉宁,邵慧慧,胡增辉,等.金鱼草AmPDS基因克隆及调节类胡萝卜素合成功能分析[J].西北植物学报,2020,40(2):193-201

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  • 在线发布日期: 2020-05-02
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