Study On The Sugar Metabolism And Cloning And Expression Of Acid Invertase In Post-harvest Pear Fruit

As the material for paper, Huangguan pear after harvested was treated by 2% calcium and coated with 1% pullulan. The research was focused on the effect of different treatments on sugar metabolism and activities of related enzymes in pear fruit during storage. Meanwhile, cloning and expression of acid invertase gene(AIV)were analyzed. It laid the foundation for the further elaboration of mechanisms of sugar metabolism and improving the quality of fruit flavor from the molecular level in pear after harvested.Chromatographic separating condition of sugars was optimized. Mobile phase was acetonitrile(HPLC grade)and ultrapure water and column temperature was 35℃, using differential detector. Mobile rate was 1.0 mL/min.Study on the physiological changes of pear fruit in different treatments showed that pullulan and calcium treatments could inhibit respiration significantly, delay the contents of titratable acid and total soluble solid decreasing, as well as the after-ripening and senescence of pear fruit. As a result, it could improve the quality of post-harvest pear.Research of the effect of different treatments on sugar metabolism and activities of related enzymes showed that pullulan treatment and calcium treatment could delay the activities of AI,NI,SS and SPS decreasing on significant level(P<0.05) or highly significant level(P<0.01), comprehensive analysis of all sensory, physical and chemical indicators, calcium treatment could be used in production and practice to maintain good storage quality of pear fruit.Changes of contents of sugars in pear fruit were caused by a variety of enzymes. The experimental results demonstrated that AI and SS were the key enzymes which regulate the sugar metabolism in pear fruit after harvested.Modified CTAB method was developed to extract total RNA from pear fruit. RNA extraction of pear was difficult for their abounding with hydroxybenzene and polysacchafide. In this study, the original CTAB method was improved. The results showed that the RNA extracted by the modified CTAB method had a good integrity and the RNA bands were clear without degradation. The ratio of the brightness of 28S RNA to that of 18S RNA was approximately 2:1 and A260/A280 was 1.913.Primers were designed according to conserved sequence of acid invertase in plant that has been published in the GenBank. The extracted RNA was reverse transcribed to cDNA, then amplified to fragment of AIV gene by PCR. The genome fragment was 429bp. The homology search of RT-PCR amplified products showed that equence homology was 95% compared with that of PsS-AIV1(BAG30919.1)in Pyrus pyrifolia and 83% compared with that of GIN1(AAB47171.1)in grape.Study on differential expressions of AIV gene showed that with the extension of storage period, expression levels of AIV gene were increased first and then decreased, and its highest expression level appeared in the third month after harvest. At the same storage period, the expression level of AIV gene in control group was lower than those of calcium and pullulan treatment groups. The results showed that pullulan and calcium treatments could delay the expression levels of AIV gene decreasing, which accorded with the changes of AI activities.