Study On The Optimization Of Genetic Transformation And Introduction Of Inorganic Pyrophosphatase Gene Into Potato

Potato (Solanum tuberosum L.) is an important crop and industrial raw materials worldwide. Dormancy and sprouting of potato tuber are very important for potato cultivation, production and processing. However, the development of potato tuber production and the processing of tuber as industrial raw materials were seriously restricted because of lacking of understanding the mechanism of tuber dormancy and sprouting and slow developing technology of artificia regulation. Therefore, it has a great significance to study the molecular mechanism and regulation of tuber dormancy and sprouting for potato cultivation, storage and industrial development.Inorganic pyrophosphatase (PPase) is a kind of hydrolase which takes inorganic pyrophosphate (PPi) as substrate. PPi also is the link of sucrose degradation in cytoplasm and plastid of potato tuber, starch synthesis and glycolysis in starch storage organ. Sucrose metabolism requires PPi at two enzymatic steps. Therefore, the characteristics of potato tuber dormancy and sprouting could be controlled by regulating the content of PPi in cytoplasm.The main purpose of the study was to construct the expression vectors of PPase gene and optimize Agrobacterium-mediated genetic transformation system of potato microtuber, and introduce PPase gene into potato cultivars for the regulation of tuber dormancy and sprouting. The main results as following:1. The prokaryotic expression vector pET-PPA was constructed by fusing potato PPase gene with vector pET-28a, and introduced into Escherichia coli BL21 (DE3). The result of SDS-PAGE analysis showed that the PPase gene could express in E. coli.2. The expression vectors pBI-SP, pBIC-SP and pBIr-SP were constructed by fusing PPase gene with the constitutive promoter CaMV 35S, tuber-specific expression promoter CIPP and the low temperature-induced promoter rd29A, respectively. The expression vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method and further proved by enzyme digestion and PCR testing3. Microtubers of two potato cultivars Gannongshu 2 and Favorita were induced with three culture methods and Agrobacterium-mediated genetic transformation system were also investigated for them. The results showed that the microtuber number induced by solid culture, solid-liquid culture and liquid culture was 4.6, 4.4, and 6.5 per flask for Gannongshu 2, and 5.4, 5.8, and 7.4 per flask for Favorita respectively. The average diameter of microtubers was 6.2 mm, 5.8 mm, and 7.7 mm for Gannongshu 2, and 6.2 mm, 5.9 mm, and 7.3 mm for Favorita. The optimum genetic transformation system was obtained as following: the bacterium concentration in value of OD600 was 0.5, the infection time was 8 min, and the co-culture time was 2 days. The optimum genetic transformation system could yield transformation efficiency of 42.6 % and 36.8 % for Gannongshu 2 and Favorita, respectively.4. Microtubers of two potato cultivars Gannongshu 2 and Favorita were used as the dornors and the expression vectors pBI-SP, pBIC-SP and pBIr-SP containing the PPase gene under the control of the constitutive promoter CaMV 35S, tuber-specific expression promoter CIPP and the low temperature-induced promoter rd29A were used as the expression vectors, respectively. Some putative transgenic potato plants were obtained by Agrobacterium-mediated transformation. PCR analysis preliminary showed that PPase gene has been integrated into the genome of potato.