The Functional Identification Of 2 Synthetic Promoters With Cold-Induced And Tuber-Specific Feature In Potato Tuber

The production of chips and fries takes a very significant part in potato precessing industry. Potato tubers are normally stored at low temperature to prevent losses from spoilage spreading, sprouting and shrinkage and to extend the processing period of industries. However, cold storage alters the carbohydrate metabolism, resulting in the accumulation of reducing sugars (RS) which usually cause unacceptable dark and bitter-tasting products at high frying temperatures owing to the reaction of the reducing sugars and the amino acid groups. Because of the complexity of metabolic pathways of RS acclimation, it is difficult to improve the processing quality in a conventional breeding and the genetic engineering have been exploited to manipulate the RS content of potato tubers in cold storage. For enhancing the transgenic efficiency and avoid possible negative impact caused by over-expression of foreign genes, it is necessary to use the promoters that are cold-inducible and tuber-specific.The main purpose of present research are to construct 2 expression vectors, pCL-900 and pCLMlb-900 which are two different synthetic promoters consisted of the same antisense AI gene, and to compare their efficiency in regulating the AI activity and the RS content of the stored tubers to deepen the knowledge of the cold-sweetening of potato tubers and its improvement. The main results obtained are as follow:1. Optimized the microtuber-disc transformation system. Through the investigation on the effects of the hormone levels, Agrobacterium concentration, and infection and co-cultivation period on the genetic transformation efficiency with, single factor experiments and then the multi-factor orthogonal test, an optimized protocol was determined as:0.4mg/L IAA, Agrobacterium concentration at OD values of 0.6,9 min. infection, and 40-48h co-cultivation.2. The pCL-900 and pCLMlb-900 had been transformed into potato separately. The regenerated plants were analyzed by PCR and showed that the two vectors were integrated into the genome of potato. Twelve and 3 transgenic lines were obtained from pCL-900 and pCLM1b-900, respectively. 3. The AI activity was inhibited at different levels by the antisense transformations driven by either of the 2 chimeric promoters. Determination of the RS content and AI activity in the transgenic tubers after cold-storage showed that the AI activity of all transgenic tubers were lower than the wild type of potato cultivar E-potato 3 (E3) except for pCL-900-7. Further analysis for the tubers stored at 4℃for 30d displayed that the AI activity of pCL-900-1, pCL-900-3, pCL-900-4, pCL-900-5, pCL-900-6, pCL-900-8, pCL-900-10, pCL-900-11, PM1B-900-1, PM1B-900-2 and PM1B-900-3 were declined at a significant level when compared with E3.4. In comparison to untransformed E3, the antisence transformation had over 30% reduction in the AI activity and 20-36% in the RS content, which were significantly correlated. After storage at 4℃for 30d, the miximum reduction of the AI activity was observed in transgenic lines pCL-900-10 and pCLMlb-900-1 which respectively had 62.1% and 58.4% decline in the activity and 51.4% and 64.8% decrease in the RS content accordantly. The results suggested that the synthetic promoters with tuber-specific and cold-inducible features can mediate the gene expression in cold stored potato tubers. Furthermore, the strategy of the antisence acid invertase can be an approach to improve the cold-sweetening of the tubers at least to a certain extent.