Constructing The Subtractive CDNA Library About The Cold Sweetening Resistance Related-genes Of Potato (S.berthaultii)

Potato (Solanum tuberosum L.) is not only the fourth food crop but also an important cash crop in the world. In order to prevent the tubers from sprouting, shrinking, pathogen infection and rotting during storage, they are usually stored under low temperature condition, which causes breakdown of starch and accumulation of reducing sugars, a complex phenomenon called low-temperature sweetening (LTS). The reducing sugar can react with free amino acids of the tubers at high temperature during frying, which is called "Maillard reaction" that results in unacceptable dark-colored and bitter-tasting products. Some wild species are tolerant to LTS. But the physical and biochemical mechanisms responsible for LTS remain unknown and genes associated to LTS-resistance and their regulation have scarcely been found. In this research, potato wild species (S. berthaultii, LTS-resistance) and cultivars (E1, LTS-sensitive) were used to construct the subtractive library to identify the genes related to LTS and establish a platform to look insight into the mechanism of LTS- resistance. The main results obtained are as following:1. Through modification of the protocol of our lab, a new total RNA isolation method for potato tubers was established by changing the content of denaturant. Using Guanidine Hydrochloride, urea and high concentration of β-Mercaptoethanol, this method efficiently solved the problems of RNA to be degraded by endogenesis RNase and the contamination of oxidated penolic compounds. High concentration of denaturant and the method of adding phenol/chloroform/isoamyl alcohol (PHI) in the thawing sample, by which restrained the starch of potato from swelling were adopted. In order to prevent from contamination of proteins and denaturant, the RNA was re-dissolved by DEPC treated water and the isolation step of PHI was added to make the RNA being free from proteins. Compared to the common method, the improved method was economical, simple and efficient. Total RNA isolated by this method was suitable for all kinds of molecular biology manipulation.2. With the method of SSH, the subtractive library CW was constructed using the tubers of wild species (CW2-1), treated at 4℃ (Tester) or 20℃ (Driver) for 5d. The subtractive library CE was constructed with the tubers of CW2-1 and E1, which were both treated at 4℃ for 5d. There were 1468 clones of CW and 1372 clones of CE that were obtained.3. The constructed libraries were preliminarily screened by the reverse Northern dot-blot. The signal intensity of the hybridizations was analyzed using the "Gel-Pro Analyzer 3.1". There were 53 clones expressed > 5× in CW and 40 clones expressed...