FvMRLKs類受體蛋白激酶在草莓果實(shí)發(fā)育中的功能及機(jī)理研究
發(fā)布時(shí)間：2018-12-15 07:57
【摘要】：果樹果實(shí)發(fā)育和成熟調(diào)控機(jī)理研究是果樹學(xué)科的核心問(wèn)題之一。然而,多年來(lái)有關(guān)果樹果實(shí)發(fā)育和成熟機(jī)理的研究基本上都限定在果實(shí)生理、生化代謝層面的探討上,而對(duì)操縱果實(shí)生理、生化代謝的更為深刻的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制幾乎沒(méi)有實(shí)質(zhì)性了解。MRLK家族(Malectin-like domain-containing receptor-like kinases)是一類含有Malectin結(jié)構(gòu)域的類受體蛋白激酶家族。因Malectin結(jié)構(gòu)域具有寡聚糖結(jié)合特性,因此推測(cè)MRLKs可能和寡糖信號(hào)轉(zhuǎn)導(dǎo)有著密切的關(guān)系。果樹果實(shí)發(fā)育和成熟過(guò)程中不可避免地發(fā)生細(xì)胞壁代謝和寡聚糖信號(hào)轉(zhuǎn)導(dǎo),因此推測(cè)MRLKs在果樹果實(shí)發(fā)育和成熟調(diào)控中可能起著重要的作用。有鑒于此,本研究以非呼吸躍變型果實(shí)草莓為材料,對(duì)MRLKs在果樹果實(shí)發(fā)育和成熟調(diào)控中的作用進(jìn)行了研究。主要結(jié)果如下:1、首先基于生物信息學(xué)分析,首次在草莓基因組中鑒定出62個(gè)MRLK家族成員,按照其在染色體上的分布位置命名為FvMRLK1-FvMRLK62。根據(jù)FvMRLK蛋白家族胞外的Malectin結(jié)構(gòu)域特征,該家族可分成6個(gè)亞家族。FvMRLK家族成員的內(nèi)含子、外顯子和Malectin保守結(jié)構(gòu)域等基因結(jié)構(gòu)和生物遺傳進(jìn)化分析表明,遺傳進(jìn)化相同的家族具有相似的結(jié)構(gòu)特征,相似的結(jié)構(gòu)決定具有相近的功能。2、基因表達(dá)譜分析表明,FvMRLK家族成員在根、莖、葉和果等各種組織中均有表達(dá),但是,根中的表達(dá)量相對(duì)高于其他組織。其中,FvMRLK家族成員中有33個(gè)成員在果實(shí)中表達(dá)。在這33個(gè)成員中,60%以上成員的基因表達(dá)特征和果實(shí)發(fā)育與成熟進(jìn)程有著密切的關(guān)系。具體來(lái)說(shuō),在果實(shí)發(fā)育的早期,表達(dá)量很高,隨著果實(shí)成熟表達(dá)水平急劇下降。進(jìn)一步分析表明,FvMRLK家族大部分成員都可對(duì)操縱草莓果實(shí)發(fā)育和成熟的內(nèi)、外因子(如激素和環(huán)境因素)產(chǎn)生靈敏的應(yīng)答反應(yīng),這些結(jié)果表明FvMRLKs在草莓果實(shí)發(fā)育和成熟調(diào)控中可能起著重要的作用。3、為了進(jìn)一步證明草莓MRLK家族在草莓果實(shí)發(fā)育和成熟調(diào)控中的作用,通過(guò)草莓果實(shí)瞬時(shí)基因操縱技術(shù),研究了草莓MRLK家族其中的一個(gè)成員,FaMRLK47,在草莓果實(shí)發(fā)育和成熟的作用。結(jié)果表明,FaMRLK47的過(guò)量表達(dá)(over-expression)可顯著抑制草莓果實(shí)的成熟,相反,FaMRKL47-RNAi沉默可促進(jìn)草莓果實(shí)成熟。與此相一致,FaMRLK47-OE可抑制一系列果實(shí)成熟標(biāo)志基因的表達(dá);相反,FaMRLK47-RNAi可促進(jìn)草莓果實(shí)成熟標(biāo)志基因的表達(dá)。該研究結(jié)果為FaMRLK家族參與草莓果實(shí)發(fā)育和成熟提供了直接證據(jù)。4、鑒于FaMRLK家族在草莓果實(shí)發(fā)育和成熟過(guò)程中的重要作用,進(jìn)一步探討了寡聚糖信號(hào)在草莓果實(shí)發(fā)育和成熟調(diào)控中的作用。成功建立了草莓果實(shí)中寡糖分離和分析體系。初步證明,寡聚糖可以作為信號(hào)在草莓果實(shí)發(fā)育和成熟調(diào)控中起著重要作用。為更加深入地探討FaMRKL家族的分子識(shí)別,研究建立了 FaMRLK47的表達(dá)和純化體系。等溫滴定微量熱技術(shù)分析初步證實(shí)FaMRLK47和寡聚糖信號(hào)具有識(shí)別特征。該研究結(jié)果為深入解析FaMRLKs信號(hào)轉(zhuǎn)導(dǎo)機(jī)理奠定了重要的基礎(chǔ)。綜上所述,本研究首次發(fā)現(xiàn)草莓MRLKs在果實(shí)發(fā)育和成熟過(guò)程中有重要的調(diào)控作用,MRLKs調(diào)控草莓果實(shí)發(fā)育和成熟可能和寡糖信號(hào)識(shí)別有著密切關(guān)系。該研究不僅為深入解析草莓果實(shí)發(fā)育和成熟機(jī)理提供了重要信息,同時(shí)對(duì)今后草莓分子育種或品質(zhì)改良也有重要意義。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S668.4
本文編號(hào)：2380295
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S668.4
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