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  本文關鍵詞： 蜻蜓鳳梨 AfGAI Western Blot 乙烯 遺傳轉化　出處：《海南大學》2017年碩士論文　論文類型：學位論文

【摘要】：蜻蜓鳳梨(Aechmea fasciata)又稱美葉光萼荷,屬于鳳梨科(Bromeliaceae)光萼荷屬(Aechmia)中一種多年生常綠草本植物。由于蜻蜓鳳梨生長期較長,生產(chǎn)中常用乙烯及其類似物催花以滿足市場要求,但是乙烯促進鳳梨開花的分子機理尚不清楚。本研究基于蜻蜓鳳梨轉錄組數(shù)據(jù)結合RACE(Rapid amplification of cDNA end)技術從蜻蜓鳳梨中克隆到一個DELLA蛋白的cDNA序列的全長,生物信息學分析后將其命名為AfGAI。AfGAI cDNA全長1960 bp,編碼序列共1764 bp,編碼587個氨基酸。對AfGAI蛋白的氨基酸序列分析,結果表明其含有DELLA蛋白家族共有的DELLA結構域和GRAS結構域,與菠蘿(Ananas comosus)、海棗(Phoenix dactylifera)、油棕(Elaeisguineensis)、巨桉(Eucalyptusgrandis)、陸地棉(Gossypium hirsutum)中的GAI同源性分別為90%、73%、73%、65%、59%。進化樹分析顯示AfGAI和菠蘿的GAI(OAY62772.1)不但同源性較高,在進化關系上也更為親近。實時熒光定量PCR檢測AfGAI表達量,結果顯示:AfGAI在18點的表達量達到最大,在6點達到最低;乙烯處理幼株后短時間內AfGAI表達量變化不大,但在24h后表達量增加;在成株中,經(jīng)乙烯處理后,AfGAI表達量持續(xù)降低,這表明AfGAI的轉錄受乙烯的調控。Western blot結果表明,外源乙烯處理能夠使AfGAI蛋白含量降低,暗示AfGAI蛋白在乙烯調控開花的過程中可能具有重要作用。利用AfGAI的CDS序列構建了 CaMV35S::AfGAI過表達載體,并利用農(nóng)桿菌介導的浸花法轉化擬南芥,獲得轉基因擬南芥陽性植株。相較于野生型和轉化了 pBI121空載體的株系,35S::AfGAI擬南芥過表達植株顯著晚花。本研究初步驗證了AfGAI的功能,揭示了乙烯通過對AfGAI的調節(jié)影響開花,有助于了解乙烯調控開花的分子機理,為進一步調控市場應用前景奠定了基礎。
[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.
【學位授予單位】：海南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S682.3

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