FvMRLKs类受体蛋白激酶在草莓果实发育中的功能及机理研究

发布时间：2018-12-15 07:57

【摘要】：果树果实发育和成熟调控机理研究是果树学科的核心问题之一。然而,多年来有关果树果实发育和成熟机理的研究基本上都限定在果实生理、生化代谢层面的探讨上,而对操纵果实生理、生化代谢的更为深刻的信号转导机制几乎没有实质性了解。MRLK家族(Malectin-like domain-containing receptor-like kinases)是一类含有Malectin结构域的类受体蛋白激酶家族。因Malectin结构域具有寡聚糖结合特性,因此推测MRLKs可能和寡糖信号转导有着密切的关系。果树果实发育和成熟过程中不可避免地发生细胞壁代谢和寡聚糖信号转导,因此推测MRLKs在果树果实发育和成熟调控中可能起着重要的作用。有鉴于此,本研究以非呼吸跃变型果实草莓为材料,对MRLKs在果树果实发育和成熟调控中的作用进行了研究。主要结果如下:1、首先基于生物信息学分析,首次在草莓基因组中鉴定出62个MRLK家族成员,按照其在染色体上的分布位置命名为FvMRLK1-FvMRLK62。根据FvMRLK蛋白家族胞外的Malectin结构域特征,该家族可分成6个亚家族。FvMRLK家族成员的内含子、外显子和Malectin保守结构域等基因结构和生物遗传进化分析表明,遗传进化相同的家族具有相似的结构特征,相似的结构决定具有相近的功能。2、基因表达谱分析表明,FvMRLK家族成员在根、茎、叶和果等各种组织中均有表达,但是,根中的表达量相对高于其他组织。其中,FvMRLK家族成员中有33个成员在果实中表达。在这33个成员中,60%以上成员的基因表达特征和果实发育与成熟进程有着密切的关系。具体来说,在果实发育的早期,表达量很高,随着果实成熟表达水平急剧下降。进一步分析表明,FvMRLK家族大部分成员都可对操纵草莓果实发育和成熟的内、外因子(如激素和环境因素)产生灵敏的应答反应,这些结果表明FvMRLKs在草莓果实发育和成熟调控中可能起着重要的作用。3、为了进一步证明草莓MRLK家族在草莓果实发育和成熟调控中的作用,通过草莓果实瞬时基因操纵技术,研究了草莓MRLK家族其中的一个成员,FaMRLK47,在草莓果实发育和成熟的作用。结果表明,FaMRLK47的过量表达(over-expression)可显著抑制草莓果实的成熟,相反,FaMRKL47-RNAi沉默可促进草莓果实成熟。与此相一致,FaMRLK47-OE可抑制一系列果实成熟标志基因的表达;相反,FaMRLK47-RNAi可促进草莓果实成熟标志基因的表达。该研究结果为FaMRLK家族参与草莓果实发育和成熟提供了直接证据。4、鉴于FaMRLK家族在草莓果实发育和成熟过程中的重要作用,进一步探讨了寡聚糖信号在草莓果实发育和成熟调控中的作用。成功建立了草莓果实中寡糖分离和分析体系。初步证明,寡聚糖可以作为信号在草莓果实发育和成熟调控中起着重要作用。为更加深入地探讨FaMRKL家族的分子识别,研究建立了 FaMRLK47的表达和纯化体系。等温滴定微量热技术分析初步证实FaMRLK47和寡聚糖信号具有识别特征。该研究结果为深入解析FaMRLKs信号转导机理奠定了重要的基础。综上所述,本研究首次发现草莓MRLKs在果实发育和成熟过程中有重要的调控作用,MRLKs调控草莓果实发育和成熟可能和寡糖信号识别有着密切关系。该研究不仅为深入解析草莓果实发育和成熟机理提供了重要信息,同时对今后草莓分子育种或品质改良也有重要意义。
[Abstract]:The research of fruit development and mature control mechanism of fruit tree is one of the core problems of fruit tree. However, the research on the mechanism of fruit development and maturation of fruit trees for many years is basically limited to the research of the physiological and biochemical metabolic level of the fruit, but the more profound signal transduction mechanism of the physiological and biochemical metabolism of the fruit is hardly known. The MRLK family (Malectin-like domain-containing receptor-like kinastes) is a family of class-like receptor protein kinases that contain the Malectin domain. Since the Malectin domain has an oligosaccharide binding property, it is presumed that the MRLKs may have a close relationship with the oligosaccharide signal transduction. In the process of fruit development and maturation of fruit tree, cell wall metabolism and oligosaccharide signal transduction are inevitable, so it is suggested that MRLKs may play an important role in fruit development and maturation regulation of fruit trees. In the light of the above, the study on the role of MRLKs in fruit development and maturation of fruit trees was studied in this paper. The main results are as follows: 1. First, based on the bioinformatics analysis, 62 MRLK family members were identified for the first time in the strawberry genome, and named FvMRLK1-FvMRLK62 according to their distribution position on the chromosome. According to the Malectin domain characteristic of the FvMRLK protein family, the family can be divided into 6 subfamilies. The genetic structure and the biogenetic evolution analysis of the intron, exon, and Malectin conserved domain of the FvMRLK family show that the same family with the same genetic evolution has similar structural characteristics and similar structural decisions have similar functions. The stem, leaf and fruit were expressed in various tissues, but the amount of expression in the root was higher than that of other tissues. Of these, 33 of the members of the FvMRLK family are expressed in the fruit. Among the 33 members, more than 60% of the members had a close relationship to the expression of the gene and the development of the fruit. In particular, in the early stage of fruit development, the expression level is high, and as the mature expression level of the fruit drops sharply. Further analysis shows that most members of the FvMRLK family can respond to the development and maturation of strawberry fruits, such as hormones and environmental factors, and these results show that FvMRLKs may play an important role in the development and maturation of strawberry fruit. In order to further demonstrate the role of the strawberry MRLK family in the development and maturation of strawberry fruit, a member of the strawberry MRLK family and the FMRLK47, a member of the strawberry MRLK family, were studied by the instant gene manipulation of strawberry fruit, and the effect of the strawberry MRLK family on the development and maturation of strawberry fruit was studied. The results showed that over-expression of FMRLK47 could significantly inhibit the ripening of strawberry fruit. In contrast, the FMRLK47-OE can inhibit the expression of a series of fruit mature marker genes; instead, the FMRLK47-RNAi can promote the expression of the mature marker gene of the strawberry fruit. The results showed that the FMRLK family had direct evidence for the development and maturation of strawberry fruit. 4. In view of the important role of the FMRLK family in the development and maturation of strawberry fruit, the role of the oligosaccharide signal in the development and maturation of strawberry fruit was further discussed. The oligosaccharide separation and analysis system in strawberry fruit was successfully established. It is proved that the oligosaccharide can play an important role in the development and maturation of strawberry fruit. In order to further study the molecular recognition of the FMRKL family, the expression and purification system of FMRLK47 was established. The analysis of the micro-thermal technology of isothermal titration showed that the FMRLK47 and the oligosaccharide signal had the characteristics of identification. The results of this study provide an important basis for the in-depth analysis of the signal transduction mechanism of the FMRLKs. In conclusion, for the first time in this study, it is found that the strawberry MRLKs plays an important role in the development and maturation of the fruit, and the MRLKrs is closely related to the development and maturation of the strawberry and the identification of the oligosaccharide signal. The research not only provides important information for deeply analyzing the development and maturation mechanism of strawberry fruit, but also has great significance to the future of strawberry molecular breeding or quality improvement.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S668.4

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