Primary Establishment Of The Spontaneous Mutant Gene Isolation System And Identification Of The Mutant Type Of The Columnar Apple (Malus Pumila)

The Columnar apple is a rare material for molecular breeding and molecular biology research. Identifying its mutant type and establishing new protocols which suit to separate spontaneous mutant gene from its genome are the prerequisites for isolating the Co gene.Wijicik and its wild type Mclntosh were taken as experimental materials to investigate new protocols for isolating spontaneous mutant gene and the mutant type of Columnar apple. The results were as follows:1. Based on the S1 nuclease and CEL I nuclease mutation detection, two new protocols were established which required no prior knowledge of the target gene and its product and suit- ed to investigate the spontaneous mutant gene. The targeted mutagenesis promoter of chlorophyll a/b binding protein was taken as the example to investigate the feasibility of the theories on the level of genome.2. S1 nuclease mutation detection system became more stable after the modification and the efficiency of detection was improved, which accorded with the requirements of functional genomics research.3. The Cytokinin accounted for the mutation. The evidences were as follows.(1) The Wijicik leaves became yellow in the dark, which suggested that it was not a brassinosteroid mutant. It can be concluded that the stem thickness was the core of mutation by the analysis of the relationship between phenotype changes and development.(2) Anatomical structure comparison showed that the cell became bigger with the its transverse elongation and without its longitudinal change, and the cell quantity increased, and the middle cortex became thicker, which was caused by columnar mutation.(3) Wijicik contains more GA3, GA4, IAA and ZR, which suggested that it was not a gibberellin mutant.4. Arabidopsis dwarf mutant was obtained by the method of RNA interference. Full-length of the cDNA sequence of apple shoot GA 20-Oxidase was amplified by RT-PCR and the sequence diversity was analyzed between Wijicik and its wild type Mclntosh. The results of RNAi indicated that the gene was related to vegetative growth and was a member of GA 20-Oxidase family. Minor expression difference of the key enzyme was found between the varieties by the method of semiquantitative PCR.