【摘要】：我國柑橘以中熟品種為主,成熟期過于集中。選育不同成熟期品種、延長鮮果的供應期有利于提高我國柑橘產(chǎn)業(yè)的競爭力。柑橘果實的成熟是一個錯綜復雜的過程,受到諸多因素如光照、溫度、激素及成熟相關眾多基因的共同調(diào)控。柑橘作為非躍變型果實,其成熟分子調(diào)控機理的研究依然較少,因此其成熟調(diào)控機理的揭示對調(diào)整現(xiàn)有品種的成熟期及柑橘產(chǎn)業(yè)的發(fā)展意義重大。本研究以奉節(jié)72-1及其晚熟芽變奉節(jié)晚橙為材料,從生理水平測定了兩者的差異,對檸檬酸含量的差異進行了分析,并對篩選到的3個果實發(fā)育相關的轉(zhuǎn)錄因子進行功能驗證。主要研究結(jié)果如下:1.果實色澤方面:測定了奉節(jié)72-1及奉節(jié)晚橙果實4個不同成熟時期,即190、208、223和248 DAF(花后天數(shù),Days after flowering)的果實色澤,發(fā)現(xiàn)190-223DAF,奉節(jié)晚橙果皮色澤H值顯著高于奉節(jié)72-1,而果肉中僅190 DAF高于奉節(jié)72-1,208-248 DAF差異不明顯。在果實品質(zhì)方面,奉節(jié)72-1可溶性固形物含量在190-208 DAF高于奉節(jié)晚橙,而可滴定酸含量在4個時期均明顯低于晚熟芽變。2.檸檬酸代謝方面:分析了奉節(jié)72-1及奉節(jié)晚橙檸檬酸含量的差異,采用qRT-PCR方法測定了果實不同發(fā)育時期檸檬酸代謝相關基因的表達。結(jié)果表明,檸檬酸含量受多種基因共同調(diào)控,與合成基因CsCS4、CsME1、CsME4、CsPEPC1等及降解相關基因CsACO1、CsGAD4、CsGS1的表達密切相關。相關性分析進一步說明,CsME1、CsME4可能是導致兩者檸檬酸含量差異的關鍵基因。3.生物信息學分析:對3個關鍵轉(zhuǎn)錄因子的蛋白質(zhì)大小、等電點、保守結(jié)構(gòu)域進行了預測,分別克隆了3個轉(zhuǎn)錄因子的啟動子區(qū)并對其順式作用結(jié)合元件進行了分析。發(fā)現(xiàn)3個轉(zhuǎn)錄因子中均含有較多激素響應元件,說明其可能通過參與多種激素信號途徑來影響果實的發(fā)育和成熟。此外,啟動子區(qū)域還包含大量與光響應相關的結(jié)合元件,說明轉(zhuǎn)錄因子通過參與光合作用或影響光信號與植物體內(nèi)多種光信號受體基因互作,從而參與調(diào)控植物多種生理代謝過程。4.轉(zhuǎn)錄因子功能驗證:克隆了3個轉(zhuǎn)錄因子的CDS區(qū)域,分別構(gòu)建了亞細胞定位和番茄超表達載體。結(jié)果表明3個轉(zhuǎn)錄因子均定位在細胞核中,屬于核蛋白。利用農(nóng)桿菌介導的遺傳轉(zhuǎn)化方法轉(zhuǎn)化番茄,陽性鑒定共得到了3株35S-DREB26、2株35S-ESE3及1株35S-bZIP9轉(zhuǎn)基因番茄。統(tǒng)計轉(zhuǎn)基因植株相關性狀發(fā)現(xiàn),超表達CsDREB26基因可以延長果實破色所需時間、降低果實著色程度;而CsESE3轉(zhuǎn)基因植株與野生型破色所需時間相近,但是超表達該基因卻明顯抑制果實著色。說明CsDREB26及CsESE3可以負調(diào)控番茄果實成熟。
[Abstract]:The main citrus varieties in China are medium mature varieties, and the maturity stage is too concentrated. Breeding different mature varieties and prolonging the supply period of fresh fruit are beneficial to improve the competitiveness of citrus industry in China. Citrus fruit ripening is a complex process, which is regulated by many factors, such as light, temperature, hormones and many genes related to maturity. As a non-jump fruit, there are still few studies on the mechanism of mature molecular regulation of citrus, so the disclosure of its ripening regulation mechanism is of great significance to adjust the maturity period of existing varieties and the development of citrus industry. In this study, Fengjie 72 / 1 and its late mature bud Changjie late orange were used as materials to determine the difference between the two at physiological level, and the difference of citric acid content was analyzed. The function of three transcription factors related to fruit development was verified. The main results are as follows: 1. Fruit color: the fruit color of Fengjie 72 脳 1 and Fengjie late orange at four different ripening stages, namely 190208223 DAF and 248 DAF (days after anthesis), was determined and 190 鈮，

本文編號：2491076