Preliminary Analysis Of The Function Of Melon Ubiquitin Ligase Gene CmRMA1H1 In Fruit Ripening

Melon cultivar Hetao honeydew?Cucumis melo L.cv.Hetao?is a dicotyledonous diploid plant of Cucurbitaceae,its fruit is a typical climacteric fruit.It is a characteristic crop in the west of Inner Mongolia,which has important economic value and nutritional value.CmRMA1H1 gene belongs to the RING type E3 ubiquitin ligase family,which can regulate plant hormone signal transduction and stress.In this paper,the CmRMA1H1 gene sequence was cloned from melon,and subcellular localization was carried out.The bioinformatics analysis showed that,there was a RAV2 transcription factor regulatory element in its promoter region,which was verified by yeast one-hybrid.The overexpression vector and CRISPR/cas9 editing vector were constructed,and melon was transformed by pollen tube pathway.Mature phenotype identification of T₁ fruit and stress tolerance analysis of T₁ seedlings were carried out respectively.The main results are as follows:?1?The cDNA sequence of CmRMA1H1 gene was cloned,and the full-length ORF of the cloned fragment was 723 bp,encoding 241 amino acids.Bioinformatics analysis showed that CmRMA1H1 protein was hydrophobic and thermally unstable,there are signal peptide cutting sites,transmembrane domains,phosphorylation sites.The protein contains a highly conserved RING finger domain.The promoter region contains abscisic acid,jasmonic acid,gibberellin,drought,ethylene and other response elements.It is speculated that CmRMA1H1 could be involved in the regulation of fruit ripening and stress response.The phylogenetic tree found that melon has the closest genetic relationship with cucumber of Cucurbitaceae,and could have similar functions.?2?pCAM-CmRMA1H1 vector was constructed and subcellular localization was analyzed.The results showed that CmRMA1H1 gene was located in endoplasmic reticulum.?3?According to the prediction of CmRMA1H1 gene promoter,it was found that the sequence contained the acting element of ethylene response factor RAV2,which was verified by yeast one-hybrid experiment.The results showed that CmRAV2 transcription factor had strong binding ability to CmRMA1H1 promoter,and CmRAV2 protein might be involved in regulating the expression of CmRMA1H1gene.?4?Detection of CmRMA1H1 gene expression in different tissues and developmental stages of melon fruit.It was found that the expression of CmRMA1H1gene was the highest in the postclimacteric stage and climacteric stage,which was about 1100 and 400 times higher than that on the 9th day after pollination,respectively.?5?The overexpression vector pPZP221-CmRMA1H1 and the genome editing expression vector pCRI-CmRMA1H1 were successfully constructed and transformed into melon.The transformed plants of T₁ generation were detected by PCR method,the positive rate of overexpression was 50%,and that of edited was 30.77%.?6?The fruit of T₁ generation transformed with overexpression vector matured about 3 days earlier than that of wild type fruit,the fruit of the transformed editing vector matured about 9-15 days later than that of the wild type.These indicated that CmRMA1H1 gene could play a role in promoting fruit ripening.According to the determination and analysis of the average weight,transverse diameter and longitudinal diameter of T₁ generation fruit,it was found that the overexpression fruit was significantly higher than that of wild type fruit,and the edited fruit was significantly lower than that of wild type fruit.It is suggested that CmRMA1H1 gene could affect the growth and development of fruit.The firmness of overexpressed fruit was significantly lower than that of wild type fruit.It is suggested that CmRMA1H1could promote fruit softening after ripening.There was no significant difference in sugar content among overexpressed fruit,edited fruit and wild type fruit.These results showed that the introduction of CmRMA1H1 gene did not affect the sugar content of the fruit.?7?The expression of CmRMA1H1 gene in positive T₁ generation fruits was analyzed.The results showed that expression of CmRMA1H1 gene in overexpressed fruit was significantly higher than that in wild type fruit,and the expression level of two fruits was about 430 times and 550 times higher than that in wild type fruit,respectively.The expression of CmRMA1H1 gene in edited fruit was significantly lower than that in wild type fruit,and the expression level of two of the fruits is about1/90 of the wild type fruit.?8?The stress tolerance of T₁ generation melon transformed plants was analyzed.It was found that CmRMA1H1 gene may increase the tolerance to salt stress and drought stress of melon seedlings.?9?The transformed plants of T₂ generation were detected by PCR method.The positive rate of overexpressed transformed plants was 61.04%,and that of edited transformed plants was 20.69%.