Identification Of ACO Gene Family And Cloning Of CmACO7and CmACO8 Gene In Melon

The melon(Cucumis melo L.)is a worldwide cultivated and consumed fruit crop in the family Cucurbitaceae which has significant potential economic value.The abundant germplasm resources as well as the available genome sequence of the melon have been released in 2012,making it an attractive model for studies on the development and ripening of fleshy fruits excepting tomato.It has been recognized that ethylene plays a important role in regulating the ripening process of climacteric fruits,and ACO is one of the rate-limiting enzyme in ethylene biosynthetic pathway.A genome-wide analysis of ACO gene family in melon was performed and candidate genes were screened for cloning based on the transcriptom data of melon.The overexpression vector and the CRISPR expression vector were transformed into the melon with ovary injection method.The results were listed as follows:1)In this study,we identified 9 A CO genes in the melon genome,and these gene members were named CmACO1-CmACO9 respectively.The 9 ACO genes were divided into two subfamilies.The amino acids homology between the encoded proteins of ACO gene family ranged from 19.95%to 75.54%.The homology of amino acid sequence in the same group was above 40%,while the homology of amino acid sequence between the two groups was only about 20%.The phylogenetic cladogram analysis of ACOs genes of melon and other species showed that CmACO1 and CsACO3,CmACO3 and CsACO2,CmACO6 and CsACO1 had the highest homology.2)According to the transcriptom data of the samples from two different periods of pulp in melon,the two differentially expressed genes were selected for qPCR detection.The result of qPCR,which showed gradually increased expression levels,was consistent with the result of transcriptome data.Gene CmACO7 and gene CmAC08 were selected as candidate genes.3)In this study,the cDNAs of CmACO7 and CmACO8 gene of the melon genome were cloned by using reverse transcription PCR.CmMACO7 had an open reading frame of 1191bp,encoding a protein with 396 amino acids,CmACO8 had an open reading frame of 1131bp,encoding 376 amino acids.The bioinformatics analysis show that CmACO7 and CmACO8 proteins are both hydrophilic proteins that contain DIOX N and 20G-Fe ?_Oxy domains.They are non-transmembrane and non-secretory proteins,as signal peptide and transmembrane structures were not found.4)The recombinant plant expression vector pPZP221-CmACO7 and pPZP221-CmAC08 were constructed by cloning the cDNA of corresponding gene into the over expression vector pPZP221.Meanwhile,the CRISPR/Cas9 expression vectors of CmAC07 and CmACO8 gene were constructed for genome editing.The expression vectors were transformed into melon with the help of ovary injection method.PCR analysis of the radicle of T1 plants showed that the positive rates were 21.5%,20.8%,10.5%and 12.3%respectively.