Identification Of Expansins Gene Family Members And Preliminary Functional Analysis Of CmEXPA8 And CmEXPA12 Genes During Fruit Development

Melon?Cucumis melo L.?,a creeping herb of the Cucurbitaceae,is a nutrient-rich fruit and an important horticultural crop.It has been considered as a model species for investigations into the regulation of fruit ripening.Expansins,widely present in various plant cell tissues,is an enzyme protein that is an important component of plant cell walls.It can promote the expansion of the cells by breaking the noncovalent bonds between cellulose and hemicellulose of cell wall,thus causing the cell wall stretching.Furthermore,expansins is also involved in cell wall softening with pH gradient-dependent.In this study,the biological function of the expansins gene during the melon fruit development process was analyzed that provides a theoretical basis for melon quality improvement and has important scientific significance.In this study,bioinformatics methods were used to identify 35 members of the expansins gene family in the melon genome.The chromosomal location of the expansins gene family was analyzed based on the physical location information in the MELONMICS database.The results showed that there's no sign of expansins genes on chromosome 5,and they were not distributed on other chromosomes evenly.The online tool GSDS software was used to draw melon exon-intron map,the analysis showed,the family members contained 1-4 introns except for the MELO3C015695T2gene;A phylogenetic tree was constructed by Using the MEGA 5.1 program with the expansins protein sequences of Cucumis melo L,Cucumis sativus L,and Arabidopsis;Melon expansion protein can be divided into four branches:EXPA,EXPB,EXLA,EXLB.The full-length cDNA of CmEXPA8 and CmEXPA12 were amplified by RT-PCR.The expression analysis of CmEXPA8 and CmEXPA12 genes showed that CmEXPA8 gene was up-regulated during fruit development,while CmEXPA12 gene was decreased along with fruit maturity.Overexpression vectors p PZP-EA8 and pPZP-EA12,CRISPR/Cas9 vectors p CRI-EA8 and pCRI-EA12 were constructed.The melon fruit was infested by transient expression with these vectors.It was observed that the site of injection with the CmEXPA8 gene CRISPR/Cas9 vector emerged green.The expression vectors were transformed into melon by pollen-tube pathway technology.The T₁ generation radicles were used as templates for PCR amplification to calculate the positive rate.The positive rates of the melons transformed with p PZP-EA8,pCRI-EA8,pPZP-EA12and p CRI-EA12 were 13.3%,16.7%,15%and 23.3%.The mature fruit phenotype of T₁ generation melons transformed with p PZP-EA8,pCRI-EA8,pPZP-EA12 and pCRI-EA12 showed that CmEXPA8 gene had accelerated the fruit ripening,while CmEXPA12 gene had promoted the enlargement of the melon fruit during the growth period.