蜻蜓凤梨AfGAI基因的克隆与功能鉴定

发布时间：2018-02-15 07:19

  本文关键词： 蜻蜓凤梨 AfGAI Western Blot 乙烯 遗传转化　出处：《海南大学》2017年硕士论文　论文类型：学位论文

【摘要】：蜻蜓凤梨(Aechmea fasciata)又称美叶光萼荷,属于凤梨科(Bromeliaceae)光萼荷属(Aechmia)中一种多年生常绿草本植物。由于蜻蜓凤梨生长期较长,生产中常用乙烯及其类似物催花以满足市场要求,但是乙烯促进凤梨开花的分子机理尚不清楚。本研究基于蜻蜓凤梨转录组数据结合RACE(Rapid amplification of cDNA end)技术从蜻蜓凤梨中克隆到一个DELLA蛋白的cDNA序列的全长,生物信息学分析后将其命名为AfGAI。AfGAI cDNA全长1960 bp,编码序列共1764 bp,编码587个氨基酸。对AfGAI蛋白的氨基酸序列分析,结果表明其含有DELLA蛋白家族共有的DELLA结构域和GRAS结构域,与菠萝(Ananas comosus)、海枣(Phoenix dactylifera)、油棕(Elaeisguineensis)、巨桉(Eucalyptusgrandis)、陆地棉(Gossypium hirsutum)中的GAI同源性分别为90%、73%、73%、65%、59%。进化树分析显示AfGAI和菠萝的GAI(OAY62772.1)不但同源性较高,在进化关系上也更为亲近。实时荧光定量PCR检测AfGAI表达量,结果显示:AfGAI在18点的表达量达到最大,在6点达到最低;乙烯处理幼株后短时间内AfGAI表达量变化不大,但在24h后表达量增加;在成株中,经乙烯处理后,AfGAI表达量持续降低,这表明AfGAI的转录受乙烯的调控。Western blot结果表明,外源乙烯处理能够使AfGAI蛋白含量降低,暗示AfGAI蛋白在乙烯调控开花的过程中可能具有重要作用。利用AfGAI的CDS序列构建了 CaMV35S::AfGAI过表达载体,并利用农杆菌介导的浸花法转化拟南芥,获得转基因拟南芥阳性植株。相较于野生型和转化了 pBI121空载体的株系,35S::AfGAI拟南芥过表达植株显著晚花。本研究初步验证了AfGAI的功能,揭示了乙烯通过对AfGAI的调节影响开花,有助于了解乙烯调控开花的分子机理,为进一步调控市场应用前景奠定了基础。
[Abstract]:The dragonfly pineapple, Aechmea fasciata, also known as Aechmea fasciata, belongs to a perennial evergreen herb of the genus Aechmia (Bromeliaceaeae). Since the growth period of pineapple is long, ethylene and its analogues are often used to promote flowers to meet the market requirements. However, the molecular mechanism of ethylene promoting the flowering of pineapple is not clear. In this study, based on the transcriptome data of dragonfly pineapple and RACE(Rapid amplification of cDNA, we cloned the full length of cDNA sequence of a DELLA protein from the pineapple. After bioinformatics analysis, it was named AfGAI.AfGAI cDNA full length 1960 BP, encoding a total of 1764 BP, encoding 587 amino acids. The results of amino acid sequence analysis of AfGAI protein showed that it contained DELLA domain and GRAS domain shared by DELLA protein family. Compared with Ananas comosus, Phoenix dactylifera, Elaeisguineensis, Eucalyptus grandiscus, Gossypium hirsutum, the homology of GAI was 90,73736559595959595.The phylogenetic tree analysis showed that AfGAI and Pineapple's GAIOAY62772.1 were not only highly homologous, but also more close to the evolutionary relationship. Real-time quantitative PCR was used to detect the expression of AfGAI. The results showed that the expression of AfGAI reached the maximum at 18:00 and reached the lowest at 6 o'clock, the expression of AfGAI changed little in a short period of time after ethylene treatment, but increased after 24 hours, while in adult plants, the expression of AfGAI decreased continuously after ethylene treatment. This indicated that the transcription of AfGAI was regulated by ethylene. Western blot results showed that exogenous ethylene treatment could reduce the content of AfGAI protein. It is suggested that AfGAI protein may play an important role in the regulation of ethylene flowering. The overexpression vector of CaMV35S::AfGAI was constructed by using the CDS sequence of AfGAI and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. Transgenic Arabidopsis positive plants were obtained. The transgenic Arabidopsis plants were significantly late in flower compared with wild type lines and transgenic plants with empty pBI121 vector. This study preliminarily verified the function of AfGAI and revealed the effect of ethylene on flowering through the regulation of AfGAI. It is helpful to understand the molecular mechanism of ethylene flowering and lay a foundation for further market regulation and application.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.3

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