Cloning, Vector Construction And Functional Analysis Of CmERFV-4 And CmERFⅢ-15 Gene In Melon

The plant hormone ethylene plays an important role in fruit ripening and senescence. It causes ethylene responses by ethylene signal transduction pathways, and causes significant changes in color, texture, flavor and aroma of fruit. Ethylene-responsive factors(ERFs), downstream component of ethylene signal transduction pathway, regulate the expression of a variety of functional genes and fruit development through identification and assoiciation with cis element GCC box of 5’upstream promoter of target genes. In this experiment, cDNA fragments of CmERFⅤ-4, CmERFⅢ-15 gene were amplified from ripening fruit of melon (Cucumis melo L. cv Hetao). The cDNA fragment length of CmERFⅤ-4, CmERFⅢ-15 were 554bp and 672bp respectively. Then, the full-length target gene were constructed into overexpression vector pPZP221 (35S-ERFⅤ-4-NOS and 35S-ERFⅢ-15-NOS), while two target gene fragments were constructed into RNAi vector pART27(pART27-ERFⅤ-4 and pART27-ERFⅢ-15). The results of real-time quantitative PCR showed that the level of expression of CmERFⅤ-4 was the highest in the root compared with other organizations, the level of expression in the fruits of CmERFⅤ-4 was highest at 40 DAP compared with other fruit development period. While the level of expression of CmERFⅢ-15 was the highest in the leaf compared with other organizations, the level of expression in the fruits of CmERFⅢ-15 was highest at 40 DAP compared with other fruit development period. Agrobacterium tumefaciens containing the overexpression vectors and RNAi vectors were injected into melon fruit by Agrobacterium-mediated transient expression technology. The injection site of the fruit emerged yellow or green phenotype after injection. The results of quantitative PCR showed that the level of expression of CmERFⅤ-4 and CmERFⅢ-15 gene increased significantly after overexpression compared with the control, exogenous introduced gene was expessed. The level of expression of the endogenous CmERFⅤ-4 was increased compared with the control after interference, However, the level of expression of the endogenous CmERFⅢ-15 was decreased compared with the control after interference, which showed that successfully inhibited the expression of the endogenous CmERFⅢ-15 gene.