Preliminary Study On The Regulation Of Melon Fruit Ripening By CmRGlase3 And CmLWRKY48 Genes

Cucumis melo L.is an important cash crop,and the research on the molecular mechanism of melon fruit development and maturation has theoretical significance and application value due to its properties and economic importance.In this study,Cucumis melo L.cv Hetao was used as material.According to the transcriptome data of melon fruits,the genes CmRGlase3 and CmLWRKY48,which were significantly differentially expressed at different fruit development stages,were selected as candidate genes.Bioinformatics analysis,cloning,expression profiling and genetic transformation studies were carried out,and the main results obtained are as follows:（1）The results showed that there were 7 members of RGlase gene family in melon,and the members of RGlase gene family were relatively conservative in evolution.RGlase gene family and WRKY gene family were constructed phylogenetic tree with the same family members in cucumber and tomato,respectively.The results showed that melon and cucumber belonged to direct line homology,and had closer phylogenetic relationship than tomato in evolution.Promoter analysis revealed that CmRGlase3 contains element o2-site related to protein metabolism,MSA-like involved in cell cycle regulation,and homeopathic element ABRE involved in abscisic acid reaction;CmLWRKY48 contains cryoresponsive element LTR,involved in abscisic acid The cis-acting element ABRE of the reaction and the cis-acting element TCA-element involved in the salicylic acid reaction.（2）The cDNA sequences of CmRGlase3 gene and CmLWRKY48 gene were amplified by RT-PCR,and the lengths were 1572 bp and 1524 bp,respectively.By real-time fluorescence quantitative PCR（RT-qPCR）,the expression level of CmRGlase3 gene in fruits was significantly higher than that in roots,stems and leaves,and reached the maximum at the climacteric stage of fruit respiration;CmLWRKY48 gene was also expressed in roots,stems and leaves,and the expression level in leaves was higher,the expression level of CmLWRKY48 gene in fruits also reached the maximum at the climacteric stage of respiration.（3）The overexpression vectors of CmRGlase3 and CmLWRKY48 genes and CRISPR/Cas9 gene editing vectors were constructed,named pPRGlase3,pPWRKY48,pYL-RGlase3 and pYL-WRKY48 respectively.Genetic transformation of melon by ovary injection.The average positive rates of PCR in T₁ generation seeds were 10.23%,9.52%,8.75%and 8.61%,respectively.In the observation of fruit phenotype of T₁ generation,it was found that the fruits of T₁ generation which were pPRGlase3 and pPWRKY48 positive,matured 6.2 and 6.0 days earlier than those of the control group,respectively.Preliminary results indicate that the CmRGlase3 and CmLWRKY48 genes have the effect of promoting melon fruit ripening.