| A series of phenylpropanoids are important metabolic products in tobacco, having important relationship with smoking security. Endogenous phenylpropanoids significantly raise smoking harmfulness and are easily inflected by external environment factors. This thesis built stable cell culture system using varieties of Honghuadajinyuan (HD) and K326 as experiment materials, studied the regulation effects of low temperature, NaCl stress and specific inhibitors of PAL and 4CL enzymes (AOA and MDCA). The results are summarized as follows:1. In sterile environments, the explant of sterile seedling was cultured under 26±2℃ and dark conditions. We screened feasible adding-concentration and matching proportion of NAA,6-BA and 2,4-D using U6’(64) uniform design. The callus inductivity was 80%(HD) and 82.5%(K326) respectively. After three subculture periods, we got stable friable calli. Gently mashed calli were cultured in MS medium without agar, and filtered by 40 μm screen mesh, then cultured in the same medium with the conditions of 26±2℃,30g/L sucrose,120rpm/min shaking and pH5.8. After three subculture periods with eight days per period, we got stable cell suspension culture system having maximum cell proliferation.2. Low temperature significantly increased coumarin content (by 92.79% in K326, by 54.04% in HD) and the enzyme activities of PAL (by 163.95% in K326, by 159.39% in HD), C4H (by 218.83% in K326, by 189.36% in HD) and 4CL (by 237.72% in K326, by 209.68% in HD) of two varieties, and significantly reduced the contents of cryptochlorogenic acid (by 69.48% in K326, by 53.53% in HD). For HD variety, low temperature significantly raised coumarin content and the enzyme activities of PAL, C4H and 4CL, significantly decreased cryptochlorogenic acid content, and had no significant influence on the contents of scopolin and chlorogenic acid. For K326 variety, low temperature significantly raised contents of coumarin and chlorogenic acid and enzyme activities. Therefore, low temperature directly improved the metabolic intensity of pathway from phenylalanine to 4-coumaric acid coenzyme A. It provides a new technology strategy in reducing smoking harmfulness and improving flavor quality.3. NaCl stress significantly promoted the enzyme activities of PAL (by 129.24% in HD, by 126.29%3. NaCl stress significantly promoted the enzyme activities of PAL (by 129.24% in HD, by 126.29%in K326), C4H (by 207.28% in HD, by 179.38% in K326) and 4CL (by 240.03% in HD, by 202.85% in K326) of two varieties and has completely inversed influence to phenylpropanoids content between two varieties. For HD variety, NaCl stress significantly increased the contents of coumarin, cryptochlorogenic acid and significantly decreased chlorogenic acid content. For K326 variety, it significantly increased chlorogenic acid content and significantly decreased the contents of scopolin and cryptochlorogenic acid. Therefore, NaCl stress raised the phenylpropanoids content difference of HD and K326 by improved cryptochlorogenic acid metabolism or chlorogenic acid metabolism.4. The experiment showed that AOA the specific inhibitor of the key enzyme PAL significantly increased the intensity of scopolin (by 43.29%) metabolism branch in HD and the intensity of chlorogenic acid (by 122.38%) metabolism branch in K326, while AOA significantly decreased PAL activities of HD (by 51.57%) and K326 (by 35.88%).The total content of phenylpropanoids respectively increased by 42.03% in HD and by 26.03% in K326. Therefore, after AOA was added, there is still another unknown metabolic pathway of coumarin accumulation.5. The experiment showed that MDCA, specific inhibitor of the key enzyme 4CL significantly increased the contents of coumarin (by 173.25% in HD, by 191.87% in K326) and chlorogenic acid (by 18.76% in HD, by 84.80% in K326), while MDCA significantly decreased 4CL activities of HD (by 33.62%) and K326 (by 32.72%). Therefore, after MDCA was added, chlorogenic acid contents were increasing because of coumarin feedback regulation. |
PDF Full Text Request