苹果柱型相关的基因筛选与鉴定

The columnar apple has compact tree form with increased spurriness and reduced branching for labor-saving cultivation and high density of planting. The columnar trait is controlled by a single dominant gene which is discovered in a bud sport from the standard apple cultivar ‘McIntosh’. Characterization of the Co gene and its genetic transformation are of great importance for breeding new cultivars with both good quality and the ideal tree form. So far, the Co has been mapped in a region that contains 64 predicted genes on the chromosome 10. In this region, there is a retrotransponson insertion in the gene desert. This insertion was speculated to up-regulate its nearby gene(s) which led to the columnar trait in apple. . In this project, the genes at Co locus which are up-regulated in ‘Wijcik’ shoot tips would be selected as candidate genes. The strategy of strict screening of candidate genes will be conducted step by step, including expression analysis of these genes in different Co phenotypes/genotypes progeny from one cross, ectopic expression in model plants, and apple genetic transformation. It will focus on: ① Correlation analysis between the differential expression of the selecte genes and the columnar index (Co phenotype and its genotype); ② How do the selected genes influence the tree form in transgenic apples when these genes are transformed into a standard cultivar ‘McIntosh’ by gene overexpression and into a columnar cultivar ‘Wijcik’ by gene silencing, respectively. ③ The mechanism of the promoter region upstream of the target gene regulating differential gene expression, and regulation of the target gene on downstream pathways. The target gene which regulates directly the columnar growth habit in apple will be finally confirmed. The project aims at verifying the nature of the Co gene and elucidating the mechanism underlying the columnar growth habit of apple..The project will provide a solid basis for further study on the causal relationship between the columnar-specific insertion at the Co locus and the level of transcription of the nearby genes, and realizing Co-specific manipulation in apple breeding.

柱型苹果树冠紧凑、短枝多、长枝少，适于果园高度密植和省力化栽培。柱型性状受单显性基因Co控制，源自普通型苹果旭的芽变，Co的鉴定和遗传转化在选育理想树型与优良品质兼具的新品种育种中很重要。目前Co定位在10号染色体上一段含有64个预测基因的区域内，其间有一个插入在基因荒漠区的反转座子，推测该插入使其附近基因上调表达，引起柱型表型。本课题以柱型苹果威赛克茎尖中上调表达的基因作为候选基因，将采用杂交群体中不同Co表现型/基因型后代分析基因表达、模式植物的异位表达和苹果遗传转化的逐步排选策略，重点研究：威赛克与旭的基因差异表达与柱型指数的关联性；基因在旭中超表达和在威赛克中沉默对转基因苹果株形的影响；启动子对基因表达差异的作用和基因对下游途径的调控。确定直接调控柱型性状的基因，实现验证Co身份的目标，解析柱型突变机制。为深入研究柱型特异插入对周围基因表达的机制和苹果育种中Co的定向操控奠定基础。

柱型苹果（columnar apple）树冠紧凑、短枝多、长枝少，适于果园高度密植和省力化栽培。柱型性状受单显性基因Co控制，源自于芽变，一个8.2kb反转录转座子插入与柱型突变有关。至今Co基因身份未知。本项目采用逐步排除候选基因策略，全面筛查Co区域内64个预测基因，确定了2个与柱型性状相关的基因。.1. 预测基因的初步甄选。通过数据库筛选、基因克隆、以及柱型和非柱型苹果茎尖中的表达分析，从64个预测基因中初步筛选了13个候选基因，其中6个上调表达和7个下调表达。.2. 候选基因复筛。利用模式植物异位表达，从13个候选基因中筛选出3个调控植株形态的基因。Co26编码2OG-Fe（Ⅱ）加氧酶，转基因植株矮化、节间缩短；Co38具有ACC-like和2OG-Fe（Ⅱ）加氧酶结构域，转基因植株分枝增加；Co41具有三十五肽重复（PPR）结构域，转基因植株叶片增大。考虑到苹果柱型性状特点，Co26和Co38是重要的候选基因。.3. 2个候选基因功能研究。Co26转基因烟草和苹果均表现出矮化、节间短、无分枝，内源赤霉素水平下降。遗传关联分析表明，柱型苹果F1群体中，Co26表达量与个体基因型和节间长度显著相关（R2=0.9186）, 柱型后代内源赤霉素明显降低。推测Co26具有降低活性赤霉素功能。.Co38转基因烟草株高正常、分枝增多，生长素转运基因PIN1、独脚金内酯合成基因CCD1、赤霉素GA2ox上调表达，推测该基因参与了复杂的激素网络。.Co26调控株高（含节间），Co38调控分枝性状，分别对应柱型苹果的矮生（含短节间）和多短枝性状。.4.Co26上下游调控基因挖掘。酵母筛库和分子互作验证，Co26与 MdMED32（RNA聚合酶调节亚基32）互作，上调表达 MdGA2ox1 和 MdGA2ox6，导致内源赤霉素含量降低。酵母单杂初步筛出与Co26启动子互作基因，可能涉及表观遗传调控。.综上所述，Co区域中筛选出了2个受8.2kb反转座子插入影响而上调表达的基因，分别对应柱型苹果的矮生（短节间）和多短枝性状。苹果柱型是一个综合性状，我们认为8.2kb反转座子就是Co基因本身，同时上调表达2个目标基因产生柱型表型。下一步工作是揭示Co区域基因调控路径，以实现树型的定向改良。