Differentially Expressed Genes During Ripening Of Persimmon Fruit

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. The ripening results in the increases in respiration and ethylene production . the changes in color and flavor, and the softening. The developmental process is regulated by multiple factors, including age, environmental signals and endogenous hormones. Cloning ripening-related genes and studying their expression patterns are important not only in theory to clarify the ripening molecular mechanism . but also in practice to control the ripening processes. Now, DDRT-PCR (differential display reverse transcription polymerase chain reaction) is one of the most widely employed techniques to identify differential expressed genes, and its application is becoming wider and wider in biological area. The experiment will evaluate the availability about DDRT-PCR in isolating differential expressed genes of ripening persimmon fruits, and gain some informations about its ripening molecular mechanismsPersimmon is rich in nutrients and has medical and health value. It is sensitive to ethylene and goes soft fastly after harvest, therefore, it is impossible to sell far away. At present, there are a lot of reports on physiological studies of ripening persimmon fruits, but a few about molecular biology.We isolated functional RNA from the ripening persimmon fruits rich in polysaccharides and polyphenolics and studied the differential expressed genes through DDRT-PCR for the first time. The differential expressed cDNAs fragments were separated on sequencing gel by silver staining, and the true differential fragments were identified via reverse Northern analysis. Finally, we obtained 15 true differential fragments, and the sequence analysis indicated that T4 fragment is highly homologous to the members of GTP binding-protein genes family. Thus, it is suggest that the ripening of persimmon fruit is triggered by enhancing ethylene signal transduction pathway, and then the expressions of ripening-related genes are regulated. The results are generalized as following.The persimmon fruits ( Kekelang) were harvested at different ripening stages, having the firmness and ethylene production measured by a GY-1 texture analyzer and gas chromatogram analyzer respectively .The results indicated that fruit firmness declined markedly and lower than 2.0 kg -cm-2 when the fruits were yellow; The ethylene production increased slowly first, the ethylene peak formed when the fruits were soften and red ,and then it declined fastly.Some methods, including TRIzoL. Tris-boric acid, Guanidinium isothiocyanate, Classic RNA extraction kit, Flash UNIQ-10 RNA extraction kit and Modified CTAB, were evaluated about their availabilities for isolating total RNA from persimmon tissue by comparing UV absorbance ratio and undenatured gel electrophoresis. The result showed that the total RNAs obtained by modified CTAB were highly pure and undegraded , The A26o/A23o ratio ranged between 1.5-2.0 and the A260/A2go between 1.7-1.9, whereas the A26o/A23o ratio were lower than 0.4 and the majority A260/A280 were below 1.5 for all other extracted RNA, indicating that the contaminations were present of polysaccharides, polyphenols and proteins.The differential expressed genes were studies through DDRT-PCR between green and turning yellow persimmon fruits with the firmness of 9.6 kg cm-2 and 5.3 kg cm-2 respectively. The total RNA samples were used for the first-strand cDNA synthesis, using the four degenerated T12MN anchored primers in the reverse transcription reactions. The RT-PCRs were performed using 104 combinations of four anchored primers and 26 random primers, Amplified cDNA were electrophoresed through a denaturing polyacrylamide gel. Finally, there were 66 differential fragments chosen from, 20 of them belonged to green fruit and 46 belonged to turning yellow fruit.The 66 differential display bands were reamplified using the same PCR conditions and primers as before. PCR products were analyzed on agarose gel and cDNAs were...