【摘要】：我國(guó)柑橘以中熟品種為主,成熟期過(guò)于集中。選育不同成熟期品種、延長(zhǎng)鮮果的供應(yīng)期有利于提高我國(guó)柑橘產(chǎn)業(yè)的競(jìng)爭(zhēng)力。柑橘果實(shí)的成熟是一個(gè)錯(cuò)綜復(fù)雜的過(guò)程,受到諸多因素如光照、溫度、激素及成熟相關(guān)眾多基因的共同調(diào)控。柑橘作為非躍變型果實(shí),其成熟分子調(diào)控機(jī)理的研究依然較少,因此其成熟調(diào)控機(jī)理的揭示對(duì)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(cè)定了兩者的差異,對(duì)檸檬酸含量的差異進(jìn)行了分析,并對(duì)篩選到的3個(gè)果實(shí)發(fā)育相關(guān)的轉(zhuǎn)錄因子進(jìn)行功能驗(yàn)證。主要研究結(jié)果如下:1.果實(shí)色澤方面:測(cè)定了奉節(jié)72-1及奉節(jié)晚橙果實(shí)4個(gè)不同成熟時(shí)期,即190、208、223和248 DAF(花后天數(shù),Days after flowering)的果實(shí)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(shí)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(gè)時(shí)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(cè)定了果實(shí)不同發(fā)育時(shí)期檸檬酸代謝相關(guān)基因的表達(dá)。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(guān)基因CsACO1、CsGAD4、CsGS1的表達(dá)密切相關(guān)。相關(guān)性分析進(jìn)一步說(shuō)明,CsME1、CsME4可能是導(dǎo)致兩者檸檬酸含量差異的關(guān)鍵基因。3.生物信息學(xué)分析:對(duì)3個(gè)關(guān)鍵轉(zhuǎn)錄因子的蛋白質(zhì)大小、等電點(diǎn)、保守結(jié)構(gòu)域進(jìn)行了預(yù)測(cè),分別克隆了3個(gè)轉(zhuǎn)錄因子的啟動(dòng)子區(qū)并對(duì)其順式作用結(jié)合元件進(jìn)行了分析。發(fā)現(xiàn)3個(gè)轉(zhuǎn)錄因子中均含有較多激素響應(yīng)元件,說(shuō)明其可能通過(guò)參與多種激素信號(hào)途徑來(lái)影響果實(shí)的發(fā)育和成熟。此外,啟動(dòng)子區(qū)域還包含大量與光響應(yīng)相關(guān)的結(jié)合元件,說(shuō)明轉(zhuǎn)錄因子通過(guò)參與光合作用或影響光信號(hào)與植物體內(nèi)多種光信號(hào)受體基因互作,從而參與調(diào)控植物多種生理代謝過(guò)程。4.轉(zhuǎn)錄因子功能驗(yàn)證:克隆了3個(gè)轉(zhuǎn)錄因子的CDS區(qū)域,分別構(gòu)建了亞細(xì)胞定位和番茄超表達(dá)載體。結(jié)果表明3個(gè)轉(zhuǎn)錄因子均定位在細(xì)胞核中,屬于核蛋白。利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化方法轉(zhuǎn)化番茄,陽(yáng)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(jì)轉(zhuǎn)基因植株相關(guān)性狀發(fā)現(xiàn),超表達(dá)CsDREB26基因可以延長(zhǎng)果實(shí)破色所需時(shí)間、降低果實(shí)著色程度;而CsESE3轉(zhuǎn)基因植株與野生型破色所需時(shí)間相近,但是超表達(dá)該基因卻明顯抑制果實(shí)著色。說(shuō)明CsDREB26及CsESE3可以負(fù)調(diào)控番茄果實(shí)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(hào)：2491076