【作者】 姚玉新；

【导师】 翟衡；

【作者基本信息】 山东农业大学 ， 果树学， 2006， 博士

【摘要】 苹果（Malus domestica B.）是世界上最重要的鲜食和加工果品之一。苹果和以苹果浓缩汁为主的加工业成为我国加入世贸组织以后最具国际市场竞争力的优势农产品之一。我国长期以鲜食苹果栽培为主,主栽品种如富士系列、元帅系列、嘎啦系列等均偏甜而少酸,导致加工产品酸度低,出口效益较低,生产上迫切需要发展适宜加工的酸度高的品种。为了研究苹果酸度调控机制,本文利用分子标记、分析代谢关键酶和cDNA-AFLP差异显示的方法对控制果实酸度的基因进行了研究。主要结果如下:1.苹果果实酸/非（低）酸性状的SSR标记分析利用苹果上开发的140对SSR引物对东光和富士的F1代群体进行了酸、非酸性状的分子标记筛选,得到一个与果实酸性状连锁的SSR标记（CH03d12）,遗传距离为8.89 cM。共显性分析表明酸（Ma）对非酸（ma）为完全显性。2.热硼酸法提取苹果果实RNA的改良在原热硼酸法的基础上加入一些提取辅助剂并根据辅助剂的特性优化提取步骤,改良后的方法可以有效去除蛋白和多糖,从而可以从不同发育阶段的苹果果实中高效、稳定的提取高质量的RNA,所得RNA的A₂₆₀/A₂₃₀大于2.0,A₂₆₀/A₂₈₀介于1.8-2.1之间。所得RNA产量高,其中前期果实RNA产率在13.40μg/g之上,后期果实RNA产率在7.02μg/g之上。3.苹果果实细胞质型苹果酸脱氢酶（cyMDH）基因的分离及其特征利用不同物种的同源序列设计简并引物,通过RT-PCR和RACE-PCR技术分离了富士苹果果实中的细胞质型苹果酸脱氢酶基因,命名为Mal-cyMDH,其全长为1,246bp,GenBank注册号为DQ221207,基因组序列分析表明编码区含有7个外显子和6个内含子。推断的蛋白序列分析表明,Mal-cyMDH大部分氨基酸区域为亲水结构,有2个推测的跨膜结构区,其中在N端第9个氨基酸下游存在一个26氨基酸的强疏水结构域,这种结构也发现于其它动植物和微生物的cyMDH中。同源性比较发现,该基因的氨基酸序列与其它植物具有90%以上的同源性,尤其是与双子叶植物的同源性更高,其中与桃子的同源性最高,达到96%;与动物和微生物的同源性也在50%以上。同时在该序列中也发现了cyMDH蛋白中高度保守的NAD结合基元TGAAGQI和催化基元“IWGNH”。这都说明本更多还原

【Abstract】 Apple （Malus domestica B.） is one of the most important processing and table fruits all over the world. Apple and its processing industry represented by concentrated juice have become one of the most competitive agricultural products in the international market after China’s accession to WTO. However, we are still facing a most outstanding problem, i.e. lacking of special processing varieties with high acidity. In order to investigate the mechanism of malic aicd accumulation in apple fruits, we conducted some researches on the genes related to apple fruit acidity by screening SSR markers, cloning and transcriptional analysis and cDNA-AFLP techniques.1. Analysis of apple fruit acid/non-acid trait by SSR markersIn this study, SSR markers linked to acid/non-acid trait in apple fruits was recruited from 140 SSR primer pairs. The screening population was composed of 91 F1 offsprings crossed by apple cultivar‘Dongguang’and‘Fuji’. Of 140 SSR primer pairs, only primer CH03d12 produced a polymorphic band linked to the acid trait with a linkage distance of 8.89 cM. The SSR marker analysis, coupled with the change of the total acid and malic acid contents, revealed that acid/non-acid trait was governed by a major gene and acid trait was complete dominant.2. Extraction of total RNA from apple flesh with the modified hot borate method.Apple fruits, especially at the riping stages, contain high contents of polysaccharides and other secondary metabolites. Therefore, it is very difficult to extract RNA from riping apple fruits. In order to resolve this problem, an modified hot borate protocol was developed for extracting high quality RNA from riping apple fruits by using assistant reagents and rearranging extraction steps. This modified method was so effective as to produce ca. 13.40μg/g total RNA from fruits at early stage, i.e., approximately 7 days after anthesis, and ca. 7.02μg/g from fruits at late stage, i.e. 7 days before harvest. The A₂₆₀/A₂₃₀ of resultant RNA was more than 2.0 as well as A₂₆₀/A₂₈₀ ranged from 1.8 to 2.1. The quality was high enough to perform RT-PCR and cDNA-AFLP analysis.更多还原