Character And Identification Of Scatophagus Argus MMP Gene Family And Their Expression In Gonads

Spotted scat（Scatophagus argus）is an important economic aquaculture fish species with commercial and ornamental values in the southeast coast of China.At present,the artificial reproduction of S.argus had been successfully implemented,but the regulation mechanism remains to be elucidated.It is well known that the gonadal development and germ cells maturation are critical for the animal reproduction,in which the molecular metabolism and extracellular matrix（ECM）remodeling were involved in preserving the gonadal structural integrity,organization,and regulation in metabolism.Matrix metalloproteinase（MMP）,a zinc-containing endopeptidase,is the key protein enzyme for ECM metabolism regulation and responsible for the ECM catabolic progress.Based on the S.argus genome and gonadal transcriptome data,the character,identification of MMP genes family,construction of phylogenetic tree,evolutionary analysis of gene synteny and collinearit were carried out.Furthermore,the mmp genes expression in S.argus gonads at different gonadal development stages and different months were detected by fluorescence quantitative PCR（q PCR）.The effects of 17β-estradiol（E₂）on gonadal mmp gene expression was analyzed by in vivo and in vitro experiment.The results were shown as follows:1.In this study,a total of 23 MMP genes were isolated and identified from the genome of S.argus,and divided into four categories:the classical MMP（mmp13,-19,-20a and-20b）,gelatinase（mmp2 and mmp9）,the membrane-type MMP(type I transmembrane-type MMP,mmp14a,-14a,-15a,-15b,-16a,-16b and-24,and glycosylphosphatidylinositol（GPI）-anchored type（mmp17a,-17b and-25）and the furin-like cleavage site MMP（mmp11,-21,-23b,-23bb and-28）.The phylogenetic tree analysis showed that MMP proteins were clustered into four categories:（1）MMP with Furin recognition site;（2）membrane-type MMP;（3）gelatinase;（4）simple structure MMP.Synteny analysis showed that the MMP gene family cluster was observed in the genome of vertebrate,and the formation of the duplicated MMP genes in teleosts arisen from its special whole-genome duplication（TS-WGD）.Gene structure analysis showed that the mmps gene structure was conservative in teleosts.2.The female and male S.argus gonadal transcriptomic analysis showed that 11mmp genes were highly expressed in gonads,including mmp2,mmp9,mmp11b,mmp14a,mmp15a,mmp15b,mmp16a,mmp17a,mmp23b,mmp24 and mmp28.By q PCR,tissue distribution analysis showed that those 11 mmp genes were mainly expressed in the kidney,spleen,pituitary and gonads etc,especially,with sex dimorphism expression in gonad and kidney.Moreover,those mmps gene expression at different gonadal development stages（phase II to IV）and different months（4 to 12month）were analyzed by q PCR.The result showed that the relative mmp28expression level in ovary was higher than that in testis,while the relative expression levels of other mmp genes in testis was higher than that in ovary.3.Male-to-female sex reversal could be induced by long term administration of exogenous 17β-estradiol（E₂）in male S.argus,with the testis transformed into the intersex ovo-testis,and finally developed into the ovary.By q PCR,the relative mmp15a,mmp15b,mmp16a,mmp17a and mmp24 expression levels in the intersex ovo-testis were significantly lower than that in testis,but showed no difference with that in ovary.In addition,the effects of E₂on gonadal mmp genes expression in testis and ovary was performed by E₂injection in vivo and incubation in vitro.After 6 h,the relative mmp2,-9,-11b and-14a expression levels were down-regulated in testis and significantly up-regulated in ovary of E₂injection group,compared with those in the control group.Whereas,after 6 h,incubation of the different concentrations of E₂in vitro could up-regulated the relative mmp2 and mmp9 expression levels in both testis and ovary.In conclusion,the character,identification,and bio-information of mmp genes family were performed in S.argus.Their expression levels analyses in gonads at different gonadal development and different months were carried out by q PCR.At the same time,the effects of 17β-estradiol on gonadal mmps gene expression was studied by in vivo injection and in vitro incubation of E₂.This study laid a foundation for further clarifying the function of S.argus mmps gene during the gonadal development and maturation.