發(fā)布時(shí)間：2018-02-15 07:19

  本文關(guān)鍵詞： 蜻蜓鳳梨 AfGAI Western Blot 乙烯 遺傳轉(zhuǎn)化　出處：《海南大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：蜻蜓鳳梨(Aechmea fasciata)又稱(chēng)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(zhǎng)期較長(zhǎng),生產(chǎn)中常用乙烯及其類(lèi)似物催花以滿(mǎn)足市場(chǎng)要求,但是乙烯促進(jìn)鳳梨開(kāi)花的分子機(jī)理尚不清楚。本研究基于蜻蜓鳳梨轉(zhuǎn)錄組數(shù)據(jù)結(jié)合RACE(Rapid amplification of cDNA end)技術(shù)從蜻蜓鳳梨中克隆到一個(gè)DELLA蛋白的cDNA序列的全長(zhǎng),生物信息學(xué)分析后將其命名為AfGAI。AfGAI cDNA全長(zhǎng)1960 bp,編碼序列共1764 bp,編碼587個(gè)氨基酸。對(duì)AfGAI蛋白的氨基酸序列分析,結(jié)果表明其含有DELLA蛋白家族共有的DELLA結(jié)構(gòu)域和GRAS結(jié)構(gòu)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(jìn)化樹(shù)分析顯示AfGAI和菠蘿的GAI(OAY62772.1)不但同源性較高,在進(jìn)化關(guān)系上也更為親近。實(shí)時(shí)熒光定量PCR檢測(cè)AfGAI表達(dá)量,結(jié)果顯示:AfGAI在18點(diǎn)的表達(dá)量達(dá)到最大,在6點(diǎn)達(dá)到最低;乙烯處理幼株后短時(shí)間內(nèi)AfGAI表達(dá)量變化不大,但在24h后表達(dá)量增加;在成株中,經(jīng)乙烯處理后,AfGAI表達(dá)量持續(xù)降低,這表明AfGAI的轉(zhuǎn)錄受乙烯的調(diào)控。Western blot結(jié)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(diào)控開(kāi)花的過(guò)程中可能具有重要作用。利用AfGAI的CDS序列構(gòu)建了 CaMV35S::AfGAI過(guò)表達(dá)載體,并利用農(nóng)桿菌介導(dǎo)的浸花法轉(zhuǎn)化擬南芥,獲得轉(zhuǎn)基因擬南芥陽(yáng)性植株。相較于野生型和轉(zhuǎn)化了 pBI121空載體的株系,35S::AfGAI擬南芥過(guò)表達(dá)植株顯著晚花。本研究初步驗(yàn)證了AfGAI的功能,揭示了乙烯通過(guò)對(duì)AfGAI的調(diào)節(jié)影響開(kāi)花,有助于了解乙烯調(diào)控開(kāi)花的分子機(jī)理,為進(jìn)一步調(diào)控市場(chǎng)應(yīng)用前景奠定了基礎(chǔ)。
[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(xué)位授予單位】：海南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S682.3

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