Identification Of Melon ACS Gene Family Members And Cloning And Genetic Transformation Of CmACS7 And CmACS11

Melon(Cucumis melo L.)is an important horticultural crop.It is a year-round oyster or climbing herb that is widely cultivated in the world.It has unique biological characteristics and important economic value.Melon is an alternative and very important model plant for the development of fleshy fruit in addition to tomato,and there are obvious changes of ethylene in the development and maturation.ACC synthase(1-aminocyclopropane-1-carboxylic acid synthase,ACS)catalyzes S-adenosylmethionine(SAM)into 1-aminocyclopropane-1-carboxylic acid(ACC)in ethylene biosynthesis.It is the key rate-limiting enzyme that determines the rate of ethylene production in plant tissue.All members of the ACS gene family were identified from genome databases using bioinformatics methods in melon(Cucumis melo L.cv.Hetao).A total of 11 melon ACS genes were identified.The analysis of the relationship between the occurrence and evolution of intron-exon subsystems showed that CmACS1 and CmACS2 belonged to type? in melon ACS genes,CmACS7,CmACS10,CmACS11 and CmACS12 belonged to type?,and CmACS3 and CmACS5 belonged to type ?.The promoter prediction results showed that the ACS gene promoter sequence mainly contained cis-acting elements involved in defense and stress responses,and cis-regulatory elements involved in response to various hormones.Semi-quantity PCR and qPCR results showed that ACS gene expression was tissue-specific,CmACS1 and CmACS11 expression levels increased during the fruit transition period,CmACS10 and CmACS12 expression levels were higher in leaves,and CmACS7 and CmACS11 were highly expressed in the ovary.The full-length cDNAs of CmACS7 and CmACS11 were cloned by RT-PCR.In addition to CmACS7 and CmACS11,the CmACS1 gene overexpression vector and CRISPR/Cas9 gene editing vector were constructed.Melon was transformed via ovary injection method,and PCR detection showed that the conversion efficiency was 12.5%-57%.By sequencing analysis,no gene editing occurred in the positive plants transformed with the editing vectors pCRI-CmACS1 and pCRI-CmACS7,and a total of 11 strains of pCRI-CmACS11,pCRI-CmACS1/11 and pCRI-CmACS7/11 transformed plants were genetically edited.