【作者】 贾海锋；

【导师】 贾文锁；

【作者基本信息】 中国农业大学 ， 果树学， 2013， 博士

【摘要】 果实发育和成熟调控机理研究一直是果树学科的一个重点问题和热点问题。多年来,有关果树果实发育和成熟调控机理的研究大部分都集中在对果实发育过程中某些生理生化代谢过程的研究。这些研究奠定了果实发育和成熟调控的基本理论基础。比如,根据果实发育和成熟过程中呼吸变化的特点,可将果实分为呼吸跃变和非跃变两类。跃变型果实认为是由植物激素乙烯调控的,而非跃变型果实的成熟机理一直不很清楚。草莓不仅是重要的果树品种之一,而且日益成为研究果树果实发育分子基础的重要材料。草莓属于非呼吸跃变型果实,最近的研究表明,植物激素脱落酸(Abscisic acid, ABA)可能在草莓果实成熟调控中起着重要的作用。但基于草莓RNA转录水平和生物信息学分析发现,蔗糖代谢和茉莉酸(Jasmonic acid, JA)代谢可能与草莓果实发育和成熟调控有着更为密切的关系。为此,本研究以草莓为试材,对蔗糖和茉莉酸在草莓果实发育和成熟调控中的作用及其机理进行了研究,主要结果和结论如下：1.随着草莓果实发育,葡萄糖、果糖和蔗糖含量都显著上升,但蔗糖含量变化和果实发育及成熟进程的关系尤为密切。外源蔗糖处理可急剧加速草莓果实的成熟,同时,蔗糖结构类似物松二糖处理也显著促进草莓果实的上色。进一步克隆了草莓基因库中7个蔗糖转运蛋白编码基因,即FaSUT1、FaSUT2、FaSUT3、FaSUT4、FaSUT5、FaSUT6和FaSUT7。研究表明,随着果实发育和成熟,FaSUT1、FaSUT2和FaSUT3表达量上升,但FaSUT1表达上升的趋势尤为剧烈,且与果实发育进程密切相关；FaSUT4、FaSUT6和FaSUT7在熟后前期表达量上升,但完全成熟时表达量又开始下降。酵母装载蔗糖速率试验表明,在所有7个蔗糖转运蛋白编码基因中,FaSUT1的蔗糖吸收转载速率最快,其次是FaSUT2。利用农杆菌介导的基因瞬时沉默和过量表达体系研究表明,FaSUT1基因过量表达可促进草莓果实的成熟；相反,FaSUT1基因沉默可延缓果实的成熟进程。与此一致,操纵FaSUT1基因的表达可操纵一系列果实成熟相关基因的表达。比如FaBG1、 FaSPS、FαAT、FaPG1、PaAI、FaQR、FaPAL和FaPT1等。重要的是,操纵FaSUT1基因的表达,可操纵ABA合成关键酶FaNCED1基因的表达,并调控果实发育过程中ABA含量的变化。这些结果表明,在草莓果实中,蔗糖不仅是重要的品质构成因素之一,而且还可以作为信号,通过操纵ABA的信号起源,在草莓果实发育和成熟调控中起着重要作用。2.在草莓果实发育初期,即从坐果到绿果中期,内源JA含量有所下降。然而从绿果中期到绿白转化期,内源JA含量急剧上升,但到果实成熟后JA含量义开始下降。因此在草莓果实发育过程中,JA含量变化趋势表明,JA可能在草莓果实成熟启动中起着重要的作用。外源MeJA处理可促进草莓果实发育和成熟进程,进一步说明JA在草莓果实发育和成熟调控中的重要作用。为深入揭示JA调控草莓果实发育和成熟的机理,在草莓果实发育中期,对JA应答基因的表达进行了RNA转录水平分析。结果发现,茉莉酸甲酯(MeJA)处理可诱导6806个基因表达量上调,其中94个基因表达量上调超过6倍,108个基因表达量上调5倍,87个基因表达量上调4倍,167个基因表达量上调3倍；共有6531个基因表达量出现了下调,89个基因表达量下调6倍以上,106个基因表达量下调5倍,86个基因表达量下调4倍,192个基因表达量下调3倍。在上调基因中,参与细胞壁代谢和色素代谢的基因数量及其变化显著,这说明JA可能主要是通过果实软化和着色操纵果实的成熟。进一步研究发现,参与JA合成的相关基因FaLOX、FaAOS、FaAOC随着草莓果实的发育其表达水平都没有发生明显的变化,而JA合成基因JFaOPDA1表达量随着果实发育和成熟,发生剧烈的变化,且这种变化与JA含量变化密切相关,说明在草莓果实中FaOPDA1可能是JA合成的关键基因。利用农杆菌介导的瞬时表达体系研究表明,FaOPDA1的过量表达可促进草莓果实上色成熟；相反,FaOPDA1的RNAi沉默可延迟草莓果实上色成熟。与此一致,基因FaOPDA1的过量表达可诱导色素代谢基因的表达,比如Chalcone Synthase (CHS)等,和果实软化基因,比如Polygalacturonase (PG)、Expansion (EXP)等。以上结果表明,JA在草莓果实发育和成熟过程中起着重要的作用。蔗糖一直被认为是结构和能源物质,而JA也一直被认为是参与抗病和抗逆的信号物质。本研究首次证实了蔗糖和JA都可作为信号物质在草莓果实发育和成熟调控中起着重要作用,揭示了草莓果实发育和成熟并非由单一信号调控,而是由多个信号系统共同操纵,信号系统间不仅存在着交义对话,还在果实发育的不同阶段可能起着不同的作用。该研究不仅加深了对非跃变型果实(至少对草莓果实)发育调控机理的认识,同时为今后果实发育和品质改良的分子调控奠定了重要的基础。更多还原

【Abstract】 The mechanism of fruit development and ripening regulation has been an important and hot issue in fruit disciplines. Over the years, many researches have been carried out on the physiological and biochemical mechanisms behind fruit development and ripening regulation. These studies had laid the basis of fruit development and ripening regualtion. According to the respiration characteristics of fruit development and ripening, fruits can be divided into climacteric fruit and non-climacteric categories. Whiel the climacteric fruit that has been well demonstrated to be controlled by the plant hormone ethylene, the machamis for the non-climacteric fruit is largely unknown. Strawberry belongs to non-climacteric fruit. Recent studies have shown that plant hormone abscisic acid (Abscisic acid, ABA) may play an important role in the regulation of strawberry fruit ripening. Neverthelss, less information is available for the deep mechims behind ABA signaling, and addiioanlly, besides ABA, whther other singals may be involved in strawbeery fruit and development is not known. Uinsg strawbeery as the research material, in the present study, strawberries as material, we have investigated the roles and mechanism of sucrose and jasmoic acid signaling in the regulation of strawbeery fruit devleopemnt and ripening. The main results and conclusions are as follows:1. With strawberry fruit development, glucose, fructose and sucrose contents were significantly increased, but changes of sucrose content and fruit development process has closer relationship. Exogenous sucrose treatment can dramatically accelerate the strawberries ripening, and sucrose structural analogues turanose treatment can also significantly promote strawberries ripening. Our further work successfully cloned7genes encoding sucrose transporter protein in strawberry fruit, namely FaSUT1, FaSUT2, FaSUT3, FaSUT4, FaSUT5, FaSUT6and FaSUT7. Studies have shown that with fruit development and ripening, FaSUTl, FaSUT2and FaSUT3expression increased, while FaSUT1gene expression increased dramatically, and is closely related with fruit development process; FaSUT4, FaSUT6and FaSUT7expression increase in pre-mature, but decrease in the fully mature fruit. Yeast loading and testing system study shows that in all seven sucrose transporter protein, sucrose uptake ratio of FaSUTl is the fastest, followed FaSUT2. Agrobacterium-mediated gene silencing and over-expression transient expression system studies showed that FaSUT1gene over-expression promotes strawberry ripening; on the contrary, FαSUTl gene silencing can delay the fruit ripening process. Consistent with this, manipulating FaSUT1gene expression can regulate a series of fruit ripening-related gene expression.Such as FaBG1, FaSPS, FaAT, FαPG1, PaAl, FaQR, FaPAL and FaPT1etc. Importantly, manipulation FaSUTl gene expression can regulate FaNCED1gene expression of a key ABA biosynthesis gene, and ABA content changes during fruit development. These results show that in the strawberry fruit, the sucrose is not only important an important factor in the composition of fruit quality, but also as a signal, through manipulating the upstream of ABA signal, plays an important role in the strawberry fruit development regulation.2. In the early strawberry fruit development, from fruit set to mid-green fruit, endogenous JA levels has decreased. Surprisingly, endogenous JA levels roses sharply from the mid-green fruit to Green and White conversion period fruit, but JA levels has begun to drop in strawberry fruit ripening. In strawberry fruit development process, unique trends of JA content mean JA may play an important role in starting strawberry fruit ripening. Exogenous JA treatment can promote strawberry fruit development and ripening process, further illustrate JA plays an important role in strawberry fruit development and ripening. In order to further reveal the mechanism of JA in regulation of strawberry fruit development and ripening, we have a RNA transcriptome analysis using mid-green fruit. The results showed that JA treatment can induce a large number of genes expression, including6806up-regulated genes,94were up-regulated more than6times,108were5times,87were4times,167were3times;6531down-regulated genes,89were down-regulated more than6times,106were5times,86were4times,192were3times. In up-regulated genes involved in cell wall metabolism and pigment metabolism genes have a significant changes, which means that JA may regulate fruit ripening through manipulation fruit softening and coloring. Further studies showed that, in many genes involved in JA bio-synthesis, such as FaLOX, FaAOS and FaAOC etc, with strawberry fruit development its expression levels have not changed significantly. However, FaOPDA1gene expression changes dramatically with fruit development its changes is related with JA content changes, implying FaOPDA1is a key enzyme of JA synthesis in strawberry fruit. Agrobacterium-mediated transient expression studies have shown that, FaOPDA1gene over-expression promotes strawberry ripening; On the contrary, FaOPDA1gene silencing delay strawberry fruit ripening. Consistent with this, FaOPDAl gene over-expression induces pigment metabolism genes expression, such as Chalcone Synthase (CHS) etc. and fruit softening genes, such as Polygalacturonase (PG), Expansion (EXP) and so on. These results above suggested that, JA plays an important role in strawberry fruit development and ripening process.3. Sucrose has been considered the structure and energy substances, and JA has been considered to be involved in disease resistance and resilience of signal substances. This is the first discovered and confirmed the sucrose and JA can be used as signal substances plays an important role in the strawberry fruit development and ripening regulation, revealing strawberry fruit development and ripening are not controlled by a single signal, but by the manipulation of multiple signaling systems together, there is a crossover between the signals, and in different fruit development stages may play a different role. This study not only greatly deepened understanding the mechanism of non-climacteric fruit development regulation (at least for strawberry fruit), in the same time, it laid an important foundation to molecular regulation of fruit development and quality improvement.更多还原