【作者】 白牡丹；

【导师】 王彩虹；

【作者基本信息】 青岛农业大学 ， 果树学， 2013， 硕士

【摘要】 苹果是世界最重要的水果之一,其栽培面积和产量在我国居于前列。进入21世纪后,苹果的发展趋于集约化、机械化和现代化,而苹果的矮化密植栽培正是体现集约化栽培和适合机械化作业的一种现代栽培方式。苹果的矮化密植具有早果丰产、易于管理和适于机械化作业等优点。柱型苹果是一种特殊的矮生突变类型,非常适合于矮化密植栽培。但目前柱型性状产生的分子机制还不清楚。研究柱型基因的分子标记,不仅在柱型性状的辅助选择方面具有重要价值,同时也对最终克隆该基因具有重要意义。赤霉素(Gibberellins, GAs)是一种重要的植物激素,具有生物活性的GAs作用于高等植物的整个生命周期。其中,对植株高矮的控制,是赤霉素最令人瞩目的生理作用之一。研究表明,植株高矮与体内的GAs类型及其合成代谢途径密切相关。目前,在苹果上,影响赤霉素合成的几个步骤的关键酶基因还不清楚,因此,克隆这些基因,对揭示其分子特征并了解其对重要性状形成的影响具有重要意义。本研究的主要内容及获得的主要结果如下：1.本研究以‘富士’x‘舞姿’的F1群体为试材,用高通量熔解曲线(HRM)方法检测到一个与苹果柱型性状紧密连锁的SSR标记(Hi01b01).对HRM反应体系的分析评价表明：采用5μL反应体积可以获得与10μL或20μL反应体积相同的检测效果；在5μL反应体积中,DNA浓度小到0.25ng/μL时,PCR扩增及随后的HRM分析效果依然良好；采用降落PCR扩增模式也能取得良好的HRM多态性检测结果。2.从‘富士’苹果茎尖中克隆到3个新的赤霉素合成过程中的关键酶基因MdCPS, MdKS和MdKAO,它们的GeneBank登录号分别为：KC433942.1、JQ281521.1和KC433941.1,编码序列长度分别为2400、2214和1512bp且编码的氨基酸残基数分别为799、737和503aa；另外,通过与苹果基因组的比对分析,将影响苹果赤霉素合成的7个关键酶基因MdCPS、MdKS、MdKO(FJ571521.1)、MdKAO. MdGA20ox1(AB037114.1)、MdGA3ox、(FJ571520.1)和MdGA2ox(FJ571521.1)分别定位在苹果第11、10、8、2、1、9和10染色体上。3.运用生物信息学的方法,对MdCPS、MdKS、MdKAO和MdGA2ox基因编码的氨基酸序列进行分析。结果显示：MdCPS和MdKS属于TPS蛋白家族,且二者均存在2个结构域；MdKAO属于细胞色素P450超家族；MdGA2ox属于依赖于2-酮戊二酸的加氧酶超家族；亚细胞定位分析发现MdCPS位于线粒体上,MdKS和MdGA2ox位于细胞质中,MdKAO位于内质网膜上；同源性分析表明,它们与已报道的其它植物的同源基因表现出较高的相似性。4.利用qRT-PCR对同一时期‘舞姿’、‘富士’及它们杂交F1代的茎尖组织中MdCPS、MdKS、MdKO、MdKAO和MdGA2ox基因的表达量进行了检测。结果表明：‘舞姿’中几个基因的表达量几乎都低于‘富士’；F1代柱型杂种群体中MdCPS和MdKS的表达量与普通型群体的差异不大；而MdKO、MdKAO和MdGA2ox的表达量明显低于普通型杂种。另外,也通过qRT-PCR对2个柱型杂种及其普通型突变体间的基因表达差异进行了分析,结果显示：柱型杂种’Z95-121’中所有基因的表达量几乎都低于它的普通型突变体‘P95-121’,这与亲本上的表现一致；而柱型杂种’Z95-177’中MdCPS、 MdKS和MdKO的表达量略高于其普通型突变体’P95-177’,MdKAO和MdGA2ox的表达量则明显低于‘P95-177’。可以推测,苹果柱型性状的形成可能与赤霉素代谢途径的部分关键酶基因的调控有关。更多还原

【Abstract】 Apple is one of the most important fruit in the world and both of its cultivation area and yield are the front in Chinese fruit production, In21st century, the development trend of apple cultivation pattern is intensive, mechanized and modernized, which can be reflected in the way of dwarfism and density planting. It has the advantage of early fruiting, high yield, easy management, and mechanized operations, and so on. Columnar apple is a special dwarf mutant and is very suitable to high density planting.So far, the molecular mechanism of apple columnar phenotype is unclear. Studies on molecular markers of columnar gene are not only valuable to the molecular assisted selection for columnar trait, but also have great meaning to map based cloning of this gene.Gibberellin (GA) is an important plant hormone. Bioactive GAs influence the entire life of higher plants. The regulation to plant height is one of the most remarkable physiological roles of GAs. It was reported that the height of plant were closely related to the type of GAs and its metabolic pathways. To date, The genes encoding the key enzymes in several steps of gibberellin biosynthesis are still unknown. Therefore, cloning these genes is of great importance to reveal their molecular characters and to understand their functions in the formation of important traits.The main researches and results were as follows:1. Using the F1population of ’Fuji’×’Telamon’as plant materials, we screened a SSR marker Hi01b01which linked to the columnar traits of apples by High resolution melting (HRM) method. The result of evaluation of different reaction systems for HRM analysis indicated that5μL reaction volume was as efficient as10μL or20μL for the polymorphism detection. In5μL reaction volume, even DNA concentration as small as0.25ng/μL was good enough for PCR amplification and the following HRM detection. It demonstrated that a touchdown PCR program could also performed very well in HRM analysis for polymorphism detection.2. In this research, the open reading frame sequences of three new genes, MdCPS, MdKS, and MdKAO were isolated from stem apical of ’Fuji’(GeneBank Accession Number is KC433942, JQ281521and KC433941, respectively). The coding sequences were2400,2214and1512bp in length and encoding799,737and503amino acids, respectively. In addition, by searching in the apple genome,7genes encoding the key enzymes of GA biosynthesis, MdCPS, MdKS, MdKO (FJ571521), MdKAO, MdGA20oxl(AB037114), MdGA3oxl(FJ571520) and MdGA2ox (FJ571521) were located on chromosome11,10,8,2,1,9and10of apple genome, respectively.3. Sequences of the amino acids encoding by MdCPS, MdKS, MdKAO and MdGA2ox were analysed by the bioinformatics. The results showed that protein MdCPS and MdKS belonged to TPS superfamily and both contained the two structural domains of TPS. MdKAO belonged to cytochrome P450superfamily. MdGA2ox belonged to the class2-oxoglutarate-dependent dioxygenases. Moreover, subcelluar location analysis showed that MdCPS was in mitochondrion; MdKS and MdGA2ox was in cytoplasm; MdKAO was in endoplasmic reticulum membrane. Homology analysis indicated that they were more identical with the homolog genes in other plants.4. Quantitative real-time PCR was used to analyze the expression of the five genes including MdCPS, MdKS, MdKO, MdKAO and MdGA2ox, in shoot apical tissue of ’Telamon’,’Fuji’and their F1progenies. The results indicated that the transcription level of almost all these genes in’Telamon’was lower than that in ’Fuji’. Moreover, as for the F1progenies, the expression difference of MdCPS and MdKS was not significant between the columnar and standard population. However, MdKO, MdKAO and MdGA2ox were distinctively lower expressed in columnar progenies than standard progenies. In addition, qRT-PCR was also employed to analyze the expression of the five genes in shoot apex of two columnar progenies and their standard mutants, The result showed that almost all genes lower expressed in columnar progeny ’Z95-121’ compared to it standard mutant ’P95-121’, which was the same as that in the columnar and standard parents. Nevertheless, the expression level of MdCPS, MdKS and MdKO in columnar progeny ’Z95-177’were slightly higher than its standard mutant ’P95-177’, but MdKAO and MdGA2ox were obviously lower expressed in the former one. Thus, we can speculated that the formation of apple columnar trait maybe related to the regulation of some genes encoding the key enzymes in the metabolic pathway of gibberellin biosynthesis.更多还原