Preliminary Functional Analyses Of CmGH18 And CmEBF Gene In Melon Fruit Ripening

The melon?Cucumis melo L.?,an important model plant for fruit ripening,is a member of the genus Cucumis in the Cucurbitaceae family.In this study,the melon variety Hetao was used as the research material to study respectively the relationship between fruit ripening and CmGH18 and CmEBF using molecular biology methods.The results are as follows:?1?According to the transcriptome database of melon fruit,CmGH18 and CmEBF that significantly differentially expressed during four stages?growth,ripening,climacteric and post-climacteric?of fruit development were selected as candidate genes.?2?The full length cDNAs of CmGH18?1097bp?and CmEBF?2134bp?,which encode 300 and 640 amino acids respectively,were cloned from the total RNA of melon fruit.The physicochemical properties,transmembrane helix region,secondary and tertiary structure,signal peptide and subcellular localization of CmGH18 and CmEBF were analyzed using bioinformatic methods.At the same time,phylogenetic analysis was conducted on these two proteins in melon and corresponding proteins in other ten species using MEGA6.0 and MEME online tools.?3?qRT-PCR was used to detect the expression pattern of CmGH18 and CmEBF genes at the different developmental stages of melon fruit.The results showed that CmGH18 exhibited the highest expression level in the fruits at 27DAP,while CmEBF had the highest expression at the stage of climacteric.?4?The overexpression vectors p PZP221-CmGH18 and pPZP221-CmEBF,and the CRISPER/Cas9 genome edited vectors pCRI-CmGH18 and pCRI-CmEBF were successfully constructed and transformed into melon via ovary injection.The positive rates of T₁ transgenic plants were determined through PCR test,which are 10%,12.5%,40%and 41.7%,respectively.The T₁ seeds from mother fruit,which possess high positive rates,were grown in the greenhouse to observe the ripening-related phenotype of T₁ melon fruits.The results suggested that CmGH18 gene accelerates fruit ripening,while CmEBF inhibits fruit ripening.