Cloning And Sequencing Analysis Of CDNA Encoding For Expansin And Its Biological Activity In Persimmon Fruit

Total RNA was extracted from Chinese persimmon fruit(Diospyros Kaki L. cv Fuping jianshi)during different development stage. Using PCR degenerate primers designed with reference to the conserved amino acid sequences of known expansins to amplify cDNA fragments by RT-PCR, two different cDNA fragments, named Cdk-exp1 and Cdk-exp2, were cloned. Changes of respiration, firmness, content of protein, biological activity of expansin in fruit after harvest were also studied in this paper. The results of this study were followed:1.To gain the higher purity and integrity total RNA, a modified hot borate method was employed. The concentration of DTT and SDS was increased 5%, the content of Proteinase K was modified to 150μl and the time of first centrifugation was postponed 10 minutes.2.The RT-PCR reaction system for persimmon fruit was established. The optimizing reaction system contained 1.5mmol/L MgCl2,0.2mmol/L dNTPs,25μmol/L Primer,0.5ng cDNA and 1.5 Unit Taq DNA polymerase.3.Two different cDNA fragments, named Cdk-exp1 and Cdk-exp2, were cloned. Cdk-exp1 and Cdk-exp2 was respectively composed of 531bp and 522bp, encoding 177 and 174 amino acids, respectively. The homologous analysis of two fragments was also accomplished.4.Biological activities of expansin during different development stage of persimmon fruit in relation to ripening and softening of fruit was studied. The result of experiment showed that expansin biological activities have a close relationship to ripening and softening of persimmon fruit.5.Expansin activities during different development stage of persimmon fruit had a higher acceleration to seedings growth in vitro of lily. The results indicated that an apparent acceleration was existent, by treated the seedings in vitro of lily with expansin crude extraction of different development stage, and the optimal concentration of expansin was 3.0g/L.