Screening, Cloning And Expression Of Genes Related To Apple Fruit Acidity

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.