sRNA参与苹果成花转变的研究

发布时间：2018-05-11 09:49

  本文选题：苹果 + sRNA　； 参考：《中国农业大学》2017年博士论文

【摘要】：苹果是我国和世界重要果树之一,为多年生木本植物。在其花诱导期内,生长点内由营养生长到生殖生长的成花转变是影响花芽数量和质量、决定下一年产量的关键环节,解析成花转变的分子机制对研究苹果生理与发育具有重要理论意义。sRNA是一类非编码的sRNA,可通过调控靶位点进而调控相关分子路径和基因网络,在植物生长发育、逆境响应、激素水平等发面发挥重要作用。目前果树中对sRNA调控成花的研究主要集中在miR156和miR172的调控机制,而对其他miRNA以及siRNA在其中的作用了解尚少;同时,在木本植物中,对sRNA合成加工路径中关键因子的功能也知之甚少。本研究以'金冠'苹果为试材,对其叶芽和花芽进行了 sRNA文库构建、测序和生物信息学分析;并克隆了sRNA合成加工路径的关键因子MdAGO1和MdAGO10,将其转化拟南芥相应突变体进行功能互补分析。主要结果如下:1通过对'金冠'苹果叶芽和花芽进行sRNA测序,发现除miR156/miR172外,还有33个差异表达的已知miRNA、6个新miRNA也参与了成花转变,其中大部分miRNA在成花转变和前人研究的苹果阶段转换中存在不同的表达模式。进一步生物信息学分析发现,差异表达miRNA主要与SPL基因、逆境响应、生长素和GA途径相关。根据靶基因分析,苹果的成花转变一方面可能在植物体内产生了相应的生理变化,诱导了相关逆境响应miRNA的差异表达,并进一步诱导了生长素相关miRNA的差异表达;另一方面,成花转变还诱导了大量siRNA在花芽中高表达,因此表明花芽中相应位点的DNA甲基化水平升高。综上所述,这些差异表达的miRNA可能通过调控SPL基因和GA路径调控营养生长,通过逆境响应路径和生长素路径调控生殖生长,siRNA则可能主要通过提高花芽内DNA甲基化水平促进成花转变。2本研究从苹果中克隆了AGO 和AGO10基因,其均具有AGO蛋白保守的PAZ和PIWI结构域,AGO10在各植物中高度保守,而AGO1则在多年生木本果树中存在特异的多次序列插入。MdAGO1和MdAGO10均主要定位于细胞核和质膜。之后我们分别构建了拟南芥AGO1和AGO10启动子驱动的MdAGO1和MdAGO10表达载体。功能互补分析发现,MdAGO1和MdAGO10可分别互补拟南芥突变体ago1-27和pnh-2的缺失表型,并使突变体中miRNA表达恢复至野生型水平。然而,与拟南芥AGO1不同,MdAGO1也可互补pnh-2的缺失表型,但未能恢复其miRNA水平至野生型。通过表型分离统计,我们再次确认了转基因植株的pnh-2背景并确定该恢复表型来自于MdAGO1的过表达。进一步分析认为,MdAGO1功能上不同于拟南芥AGO1的原因可能来自于其蛋白N端插入的多年生木本果树特异序列。
[Abstract]:Apple is one of the most important fruit trees in China and the world, and it is a perennial woody plant. During the flower induction period, the floral transformation from vegetative growth to reproductive growth in the growing point is the key link that affects the number and quality of flower buds and determines the yield of the next year. The molecular mechanism of floral transformation is of great theoretical significance for the study of apple physiology and development. SRNA is a kind of non-coding sRNAs, which can regulate the molecular pathways and gene networks by regulating the target sites, and respond to plant growth and stress. Hormone levels and other hair surface play an important role. At present, the studies on the regulation of flower formation by sRNA in fruit trees are mainly focused on the regulatory mechanisms of miR156 and miR172, but little is known about the role of other miRNA and siRNA in them, meanwhile, in woody plants, Little is known about the function of key factors in the sRNA synthesis path. In this study, the leaf buds and flower buds of 'Golden Crown' apple were constructed by sRNA library, sequenced and analyzed by bioinformatics. MdAGO1 and MdAGO10, the key factors of sRNA synthesis pathway, were cloned and transformed into Arabidopsis thaliana corresponding mutants for functional complementation analysis. The main results were as follows: 1 by sequencing the sRNA of the leaf buds and flower buds of 'Golden Crown' apple, we found that in addition to miR156/miR172, there were 33 known miRNAs differentially expressed, and six new miRNA were also involved in the flower formation transition. Most of the miRNA have different expression patterns in flowering transition and apple stage transition. Further bioinformatics analysis showed that differential expression of miRNA was mainly related to SPL gene, stress response, auxin and GA pathway. According to the target gene analysis, on the one hand, the flowering transformation of apple may produce corresponding physiological changes in the plant, induce the differential expression of miRNA in response to related stresses, and further induce the differential expression of auxin related miRNA in the plant, on the other hand, Floral transformation also induced a large number of siRNA overexpression in flower bud, which indicated that the DNA methylation level of the corresponding site in flower bud was increased. In conclusion, these differentially expressed miRNA may regulate vegetative growth by regulating SPL gene and GA pathway. Regulation of reproductive growth by stress response pathway and auxin pathway may promote floral transformation by increasing the level of DNA methylation in flower buds. 2. In this study, AGO and AGO10 genes were cloned from apple. The conserved PAZ and PIWI domain AGO10 of AGO protein were highly conserved in all plants, while AGO1 had specific multiple sequence insertions in perennial woody fruit trees. MdAGO1 and MdAGO10 were mainly located in nucleus and plasma membrane. Then we constructed Arabidopsis thaliana AGO1 and AGO10 promoter driven MdAGO1 and MdAGO10 expression vectors. Functional complementation analysis showed that MdAGO1 and MdAGO10 could complement the deletion phenotypes of ago1-27 and pnh-2 in Arabidopsis thaliana mutant, and restore miRNA expression to wild-type level. However, unlike Arabidopsis thaliana AGO1, MdAGO1 could complement the deletion phenotype of pnh-2, but could not recover its miRNA level to wild-type. By phenotypic isolation statistics, we reconfirmed the pnh-2 background of transgenic plants and confirmed that the recovery phenotype was derived from overexpression of MdAGO1. It is suggested that the reason why MdAGO1 is functionally different from Arabidopsis AGO1 may be due to the specific sequence of perennial woody fruit trees with N-terminal insertion of MdAGO1 from Arabidopsis thaliana.
【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】：S661.1

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