Exogenous CpTI Gene Expression In Fruit And Its Genetic Analysis In Gala Apple

The behavior of foreign genes in the progeny of transgenic plants plays an important role in the aspect of transgene application. The laboratory successfully transferred CpTI insect-resistant gene into Gala apples, and the transgenic lines were planted in 1999 at the Agricultural University specimen garden. The research focused on the characteristics of fruits sampled either from the transgenic gala apple trees or from the Fuji trees that pollinated by the transgenic gala. The main results were as following:1. Modified CTAB method, SDS method, and the high salt and low pH SDS method were used for apple seeds and pulps total genomic DNA extraction. The results showed that, for the genomic DNA extracted from seeds, the three kinds of extraction methods could obtain high purity DNA, but DNA yield was quite different, the modified CTAB method of DNA obtained the highest yield with 0.221 mg / g fresh weight, and the SDS method and the high salt and low pH SDS method obtained 0.053 mg / g fresh weight and 0.034 mg / g fresh weight DNA, respectively. For the extraction of genomic DNA from the flesh, the SDS method gave the best purity and yield with DNA of the A260 / A280 value at 1.808 and yield at 0.012 mg / g fresh weight, while the other two methods for extraction showed poor quality. Therefore, the improved CTAB method was more suitable for extraction of seed DNA, while the SDS method was good for extracting fruit DNA.2. PCR analysis of CpTI gene behaving in seeds showed that the positive to negative ratio was 1.85:1 and 0.68:1 for the transgenic gala apple pollinated by wild type Fuji apple and for the non-transgenic Fuji apple pollinated by the transgenic gala, respectively. Both met the 1:1 Mendelian segregation test by X2 analysis.3. With 50 mg / L Kan resistance screening test, the ratio of positive to negative plantlets that generated from the seeds of transgenic gala apple was 1.6:1, and that did not meet the 1:1 Mendelian segregation test by X2 analysis. The ratio of positive to negative plantlets that generated from the seeds of non-transgenic Fuji apple pollinated by the transgenic gala was 0.6:1, and that met the 1:1 Mendelian segregation test by X2 analysis. Different seeds from the same fruit showed gene segregation, but no regular pattern was found.4. Pollen DNA extracted from the transgenic gala was analyzed with PCR technic, and foreign CpTI gene was found.