菘蓝IiEMF基因的克隆与功能分析
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国家自然科学基金(31171486);


Cloning and Functional Analysis of IiEMF Gene in Isatis indigotica Fort.
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    摘要:

    该研究以菘蓝(Isatis indigotica Fort.)转录组数据为基础,克隆得到菘蓝EMF基因的cDNA全长,命名为IiEMF。(1)序列分析表明,IiEMF基因开放阅读框长度为1 896 bp,编码631个氨基酸。进化树分析表明,菘蓝IiEMF蛋白与甘蓝(Brassica oleracea)EMF蛋白亲缘关系最为接近。(2)实时定量PCR结果显示,IiEMF在菘蓝不同器官中均有表达,且在叶中表达量最高,果实中表达量最低; IiEMF基因在菘蓝抽薹开花过程中叶内的表达量呈先升后降的趋势,并于初花期表达量达到最高后逐渐降低回落;在花/果期IiEMF基因表达量较花蕾中明显降低。(3)成功构建了超表达载体pCAMBIA1300EMF,经农杆菌介导侵染拟南芥,PCR鉴定表明,有7株为超表达转IiEMF基因植株。(4)表型观察发现, 在长日照和短日照条件下,与野生型相比2个转IiEMF基因拟南芥株系的开花时间都明显较早(提前6~10 d),且转IiEMF基因株系的莲座叶数比野生型多10片以上,叶片也比野生型大而肥厚。(5)qRTPCR检测结果显示,在拟南芥营养生长过程中,过表达IiEMF显著抑制了拟南芥AtAP1、AtCOAtLFY的表达,而促进了AtFLC的表达;当拟南芥开花时,转基因株系中的AtAP1和AtFLC表达量均高于野生型,AtCOAtLFY的表达量显著低于野生型。研究表明,过量表达IiEMF基因能够促使拟南芥提前开花,且IiEMF可能是通过影响多种开花途径来共同调节促进拟南芥的早花。

    Abstract:

    In this study, the IiEMF gene was first cloned based on the Isatis indigotica transcriptomic analysis. (1) Sequence analysis showed that the IiEMF gene contains an open reading frame of 1 896 bp encoding 631 amino acids. The phylogenetic analysis suggests that the IiEMF protein was relatively close to the EMF protein of Brassica oleracea. (2) Realtime quantity PCR (QPCR) resulted that IiEMF existed in all organs in I. indigotica. The highest expression level was observed in leaves and the lowest was in fruits. IiEMF expression level in the leaves increased first and then decreased and the highest peak emerged at first flower appearing stage. The IiEMF expression level in flower/fruit stage was significantly lower than that in flower bud stage. (3) The overexpression vector pCAMBIA1300EMF was constructed and then was introduced into A. thaliana by Agrobacterium. There are 7 strains of A. thaliana plants of transgenic EMF gene detected by PCR. (4) The results of phenotype observation indicated that the flowering time of the two transgenic EMF arabidopsis strains was significantly earlier than that of the wild type (6~10 d) under longday and shortday conditions. The rosette leave number of transgenic IiEMF gene lines was at least ten more than that of the wild type, and the leaves were also larger and thicker than that of wild type. (5) QPCR resulted that overexpression of IiEMF significantly inhibited the expression of AtAP1, AtCO and AtLFY and promoted the expression of AtFLC in the vegetative growth process of A. thaliana. The expression levels of AtAP1 and AtFLC in transgenic IiEMF gene lines were higher than that of wild type, and the expression levels of AtCO and AtLFY were significantly lower than that of wild type in reproductive growth period of A. thaliana. This study showed that overexpression of IiEMF promoted A. thaliana flowering and IiEMF may regulate A. thaliana flowering time by influencing multiple flowering pathways.

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白 钰,唐晓清,王芳权,等.菘蓝IiEMF基因的克隆与功能分析[J].西北植物学报,2020,40(1):1-9

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  • 在线发布日期: 2020-04-02
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