Cloning And Expression Analysis Of ACS Gene Family In Cucurbita Maxima

Ethylene is an important plant hormone on sex determination.Therefore,the relationship between ethylene biosynthesis,signal transduction and sex differentiation in cucurbita crops has been the focus research.At present,most studies have focused on isolation,cloning,and functional of genes involved in ethylene biosynthesis and signal transduction pathways in cucumber and melon.There are relatively few reports on critical gene involved in ethylene synthesis and signal transduction pathways in pumpkin.In this study,the ACC synthase gene(ACS gene)was cloned from the genomic DNA of subgynoecious inbred lines ‘2013-12’ and androecious inbred lines ‘9-6’ of Cucurbita maxima according to the published C.maxima genome sequence information.The expression patterns of the ACS family genes in different tissues,organs,and flower sites in two stains preliminarily predicted their functions and provided theoretical basis for further research on the molecular regulation mechanism of sex differentiation in pumpkins.The main findings were as follows:(1)A total of 13 Cucurbita maxima ACS genes were searched in published C.maxima database,and named according to the sequence of their gene ID.The ACS gene family were grouped and bioinformatics analysisof them were produced.The results showed that the members of this gene family have differences in conserved domain sequences,chromosome location,gene structure,and physicochemical properties,which may make these family member genes perform different functions.(2)CmaACS1,CmaACS4,CmaACS5,CmaACS7,CmaACS9 and CmaACS10 of ACS family were successfully cloned from C.maxima in line ‘2013-12’ and line ‘9-6’,respectively.The open reading frame of the cloned fragments ranged from 1257 to 1455 bp in length,encoding418 to 484 amino acids.(3)The functions of ACS genes were preliminary investigate using qRT-PCR performance in line ‘2013-12’ and line ‘9-6’.As the result of expression patterns in different tissues,four genes CmaACS1,CmaACS4,CmaACS7,and CmaACS9 were specifically expressed in male and female flowers.Then,the functions of four genes were presumed with gene expression in different flowering stages,floral organs,and different agents.CmaACS1 could not involved in sex determination in pumpkin.The expression of CmaACS1 in petals gradually increased with the development of petals,which indicated it may be involved in development of petals..the expression of CmaACS4 increased in the late period of stamen and pistil development,which declared that CmaACS4 might involve in flower later development.CmaACS7 showed highestexpressionin female flower at S1 stage specifically in flower development stages,and the expressionin of female flower is higher than male flower,which indicated that CmaACS7 might be involved in sex determination,and high expression could inhibit stamen development effectively.CmaACS9 showed highest expressionin male flower at S1 stage specifically in flower development stages,and the expression was significant decrease under ethylene treatment.It indicated that CmaACS9 might be involved in sex determination,and high expression might inhibit pistil development effectively.