Genome-wide Analysis Of ACS Family In Cucumber(Cucumis Sativus L.)and Functional Identification Of CsACS2 In Sex Determination

Due to its diversity in sex types,cucumber(Cucumis sativus L.)has become a model plant for sex-determination research in plant.Three major genes—F/f,M/m,and A/a—regulate the sex types in cucumber plant.All of them have been cloned recently,and they all encoded homologies of 1-aminocyclopropane-1-carboxylate synthase(ACS).As a result,the genome-wide identification of cucumber ACS family members is essential for research of mechanisms of sex determination in cucumber.In this study,the cucumber ACS family members were identified by using cucumber genome database.We performed bioinformatics analysis and expression anlysis of cucumber ACS family.As a result,we isolated 8 members of ACS family.To study the function of CsACS2,with the organ-specifc promoter PAP3,we transformed the ethylene synthesis cucumber gene CsACS2 to generate transgenic Arabidopsis plants,The main results are as follow:1.The Hidden Markow Model(HMM)profile of the Aminotransferase class I and II domain(Pfam: PF00155)was used as a blast query against the cucumber genome database.Finally,8 members of cucumber ACS family were identified and used to perform further bioinformatics analysis.Among them,CsACS1(Csa6G496450),Cs ACS2(Csa1G580750),Cs ACS4(Csa6G006800)and CsACS11(Csa2G353460)have been reported.CsACS5(Csa4G099220),CsACS6(Csa4G049610),Cs ACS8(Csa3G177920),CsACS9(Csa5G157380)were reported for first time.2.The expression analysis of ACS gene family in different tissues from different cucumber lines showed that CsACS4,CsACS1,CsACS2,CsACS11,Cs ACS8 and CsACS9 were up-regulated in shoot apices from Gy14 line compared with the gene expression level in roots.CsACS6 was up-regulated in stems from Gy14 line;CsACS5 was up-regulated in young leaves from Gy14 line;While in 9930 line,compared with the gene expression level in roots,CsACS4 was up-regulated both in stems and shoot apices and the up-regulation was higher in stems;CsACS5,CsACS and CsACS1 were not up-regulated in stems,young leaves,shoot apices from 9930 line;CsACS11 was up-regulated in shoot apices from 9930 line;Cs ACS6 and CsACS8 were up-regulated in young leaves from 9930 line;CsACS9 was up-regulated in young leaves and shoot apices and up-regulation in young leaves was higher.While,in H34 line,compared with the gene expression level in roots,all ACS genes were not up-regulated in stems,young leaves and shoot apices.3.We transformed the ethylene synthesis cucumber gene CsACS2 to generate transgenic Arabidopsis,driven by the organ-specific promoter PAP3.We evaluated CsACS2,AtAP3,AtPI gene expression in floral buds younger than stage 10 from transgenic Arabidopsis plants with PAP3::CsACS2-GUS which were verified via PCR.The results showed that CsACS2 gene expression is higher than Arabidopsis wild type.While AtAP3,AtPI gene expression are lower than Arabidopsis wild type.The histochemical staining of young floral buds from transgenic Arabidopsis with PAP3::CsACS2-GUS indicated that CsACS2 has specific expression in stamen.A dissecting microscope and SEM were employed to conducted morphologic observation of transgenic Arabidopsis and Arabidopsis wild type.The results showed that transgenic Arabidopsis plants with PAP3::CsACS2-GUS were male sterile and no silique set.And transgenic Arabidopsis plants with PAP3::CsACS2-GUS included three unnormal types of stamen morphology,which were stamen topped by papillar structure,carpelloid stamen,filamentous stamen.We found that stamen-specific expression of CsACS2 significantly affected stamen.