奉节72-1及其晚熟芽变果实柠檬酸代谢相关基因表达分析及转录因子CsDREB26、CsESE3和CsbZIP9功能验证
发布时间：2019-06-02 11:50
【摘要】：我国柑橘以中熟品种为主,成熟期过于集中。选育不同成熟期品种、延长鲜果的供应期有利于提高我国柑橘产业的竞争力。柑橘果实的成熟是一个错综复杂的过程,受到诸多因素如光照、温度、激素及成熟相关众多基因的共同调控。柑橘作为非跃变型果实,其成熟分子调控机理的研究依然较少,因此其成熟调控机理的揭示对调整现有品种的成熟期及柑橘产业的发展意义重大。本研究以奉节72-1及其晚熟芽变奉节晚橙为材料,从生理水平测定了两者的差异,对柠檬酸含量的差异进行了分析,并对筛选到的3个果实发育相关的转录因子进行功能验证。主要研究结果如下:1.果实色泽方面:测定了奉节72-1及奉节晚橙果实4个不同成熟时期,即190、208、223和248 DAF(花后天数,Days after flowering)的果实色泽,发现190-223DAF,奉节晚橙果皮色泽H值显著高于奉节72-1,而果肉中仅190 DAF高于奉节72-1,208-248 DAF差异不明显。在果实品质方面,奉节72-1可溶性固形物含量在190-208 DAF高于奉节晚橙,而可滴定酸含量在4个时期均明显低于晚熟芽变。2.柠檬酸代谢方面:分析了奉节72-1及奉节晚橙柠檬酸含量的差异,采用qRT-PCR方法测定了果实不同发育时期柠檬酸代谢相关基因的表达。结果表明,柠檬酸含量受多种基因共同调控,与合成基因CsCS4、CsME1、CsME4、CsPEPC1等及降解相关基因CsACO1、CsGAD4、CsGS1的表达密切相关。相关性分析进一步说明,CsME1、CsME4可能是导致两者柠檬酸含量差异的关键基因。3.生物信息学分析:对3个关键转录因子的蛋白质大小、等电点、保守结构域进行了预测,分别克隆了3个转录因子的启动子区并对其顺式作用结合元件进行了分析。发现3个转录因子中均含有较多激素响应元件,说明其可能通过参与多种激素信号途径来影响果实的发育和成熟。此外,启动子区域还包含大量与光响应相关的结合元件,说明转录因子通过参与光合作用或影响光信号与植物体内多种光信号受体基因互作,从而参与调控植物多种生理代谢过程。4.转录因子功能验证:克隆了3个转录因子的CDS区域,分别构建了亚细胞定位和番茄超表达载体。结果表明3个转录因子均定位在细胞核中,属于核蛋白。利用农杆菌介导的遗传转化方法转化番茄,阳性鉴定共得到了3株35S-DREB26、2株35S-ESE3及1株35S-bZIP9转基因番茄。统计转基因植株相关性状发现,超表达CsDREB26基因可以延长果实破色所需时间、降低果实着色程度;而CsESE3转基因植株与野生型破色所需时间相近,但是超表达该基因却明显抑制果实着色。说明CsDREB26及CsESE3可以负调控番茄果实成熟。
[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，
本文编号：2491076
[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，
本文编号：2491076
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