Identification Of The Differential Expression Of MRNA During Maturation Of Papaya Fruits

Papaya,as a well-known tropical and subtropical fruit,is esteemed for its and abundant nutrition and flavorsome. Papaya is a climacteric fruit very susceptible to significant post-harvest loss due to fast softening and being easy to decay during post-harvest storage. Gene regulation play a very important role in Papaya fruit ripening, so investigation of gene expression difference in Papaya fruit ripening process can help us to understand the gene expression pattern which regulate Papaya fruit ripening and lay a foundation for further understanding the gene regulation mechalism of Papaya fruits ripening.In our previous research, we have obtained a batch of differential expression genes which are related to post-harvest ripening of Papaya. On this basis, in this study, we selected eight of them to be verified and analyzed by Real-time PCR. The eight genes include glu(glutathione-transferase),1671(Pirin Protein),1691 (Papaya chloroplast genome),1711(16S ribosome RNA), cy(Ychromosome-sequence),1678(uridine-cytidine kinase),1689(metal tolerance protein),,and 1729(L-threonine aldolase). In this study, red-fleshed Sunrise solo Papaya was used to analyze the temporal and spatial expression of these eight genes from different tissue and at different stage of fruit development and ripening. Results show that expression of eight genes at transcription levels can be detected in flowers, leaves and fruits, but the expression level of eight genes in different tissue and at different fruit development and ripening stage is significantly different. During Papaya fruit development and ripening process, the expression of glu, cy,1671,1678,1691, and 1689 genes increased. The expression pattern of 1711 gene is not significant, it increased in the early stage of fruit ripening but either increased or decreased in the mid and late stage of fruit ripening (The expression level is 1.0FRn-3.1FRn). The expression level of 1729 is lower in ripen fruits than in unripe fruits during Papaya fruit development and ripening process, and it decreased in the early stage of fruit development and ripening, but maintain e stable in the mid and late stage of fruit development and ripening. glu gene features the largest difference of expression level. The expression difference between ripen fruits and unripe fruits is more than 600 fold. The 1671 gene expression level is more than 200 times in ripen fruits higher than in unripe fruits. While 1729 gene expression level maintain stable during the whole Papaya fruit development and ripening, the expression difference is 0.5-1 fold.By using electronic cloning technique combined with PCR, we obtained the full-length sequences of 1729,1678,1671,1689 and submitted them to GenBank. The accession numbers are HM068864, HM068867, HM068865 and HM068866.The full length of 1729 gene of Papaya is 1121bp.1729 protein is highly conservative, it has the highest amino acid(aa) sequence similarity with 1729 protein in castor-oil plant, reaching 82%, followed by potato, reaching 76. The Phylogenetic tree analysis of 1729 indicates that Papaya 1729 is most closely related to potato, followed by castor-oil plant. Papaya 1678 has a full-length of 1408 bp.1678 protein is highly conservative too, it has the highest aa sequence similarity with 1678 protein in castor-oil plant, trichocarpa and Arabidopsis, reaching 78%. The Phylogenetic tree analysis of 1678 shows that Papaya 1678 is most closely related to grain sorghum, followed by Arabidopsis. Papaya 1671 has a full-lenth of 913bp,1671 protein is also highly conservative, it has the highest aa. sequence similarity with 1671 protein in soybean, reaching 86%, followed by grape and medicago truncatula, the silmilarity are 84%and82% respectively. The Phylogenetic tree analysis of 1671 shows that Papaya 1671 is most closely related to grape, followed by rice. Papaya 1689 has a full-lenth of 1237bp,1689 protein is also highly conservative, it has the highest aa sequence similarity with 1689 protein in grape, reaching 86%, followed by soybean and castor-oil plant, the silmilarity are 75%. The Phylogenetic tree analysis of 1689 indicates that Papaya 1689 is most closely related to grape, followed by rice.Our study indicates that the expression of glu,cy,167,1678,169,1689 genes increased with the ripening of Papaya fruit, which suggest that these genes play a important role in regulating the Papaya fruit ripening process, but how these genes regulate Papaya fruit ripening is still needed to further explored.