Cloning And Analysis Of Several Fruit Ripening Genes From Papaya (Carica Papaya L.)

Papaya fruit undergoes a postharvest ripening process, which plays an important role in improving the quality and extending the shelf life of papayas. To research and manipulate postharvest papaya ripening, a better understanding of the mechanism of postharvest ripening is necessary. It is a main way to isolate differentially expressed genes at the stage of postharvest papaya ripening by cDNA-AFLP technique. This study was aimed at the cDNA fragment which relate to plant growth, and the cloning of corresponding full length sequences, base on the differentially expressed genes from papya.Papaya WD-repeat protein gene and Zinc finger protein gene were cloned by RACE.Semi-quantitative RT-PCR method was used to study these genes expression in different stages of papaya fruit ripening. The results showed that two genes were expressed in the four stages of fruit, and the expression changed with the corresponding of the fruit ripening, indicating that the genes related to the fruit ripening. The major results obtained from the study are summarized as fellow:1 Cloning and analysis of WD-repeat protein gene from papayaWD-repeat protein gene was cloned by rapid amplification of cDNA ends from papaya. The full length cDNA of WD-repeat protein gene was 1091bp with an opening reading frame of 780bp encoding a protein with 259 amino acids. It contains a 5′untranslated region of 223 bp and a 3′flanking sequence of 227 bp. The deduced amino acid sequence of WD-repeat protein gene was compared with the sequences of other higher plants such as Populus trichocarpa, Medicago truncatula, Vitis vinifera, and so on, and the homology is 70% and above.This means WD-repeat protein gene has a high conservatism in higher plants. And constructed phylogenetic tree using WD-repeat protein gene from different species .The result showed that WD-repeat protein gene of papaya and Vitis vinifera had the minimum distance of the evolution that meant they had the closest relationship.2 Cloning and analysis of Zinc finger protein gene from papayaThe full length cDNA of Zinc finger protein gene was 1378bp and it was obtained by rapid amplification of cDNA ends (RACE) from papaya.It contains a largest ORF that is 1023bp encoding 340 amino acids. The deduced amino acid sequence of Zinc finger protein gene was compared with the sequences of other higher plants such as Arabidopsis thaliana, Vitis vinifera, Populus trichocarpa, and so on, and the homology is 51% and above. And constructed phylogenetic tree using Zinc finger protein gene from different species .The result showed that Zinc finger protein gene of papaya and Vitis vinifera had the minimum distance of the evolution that meant they had the closest relationship.3 Analyses by RT-PCRActin gene as an internal gene, Semi-quantitative RT-PCR method was used to study WD-repeat protein gene and zinc finger protein genes expression in different stages of papaya fruit ripening. The result showed that the expression of WD-repeat protein gene and zinc finger gene were increased gradually with papaya fruit maturing and decreased when fruit began to soften.This shows that these genes have certain relevant to papaya maturity and the two genes may have some synergy.