Molecular Cloning,Expression Patterns And Function Exploration Of FOXL2 In Macrobrachium Rosenbergii

Macrobrachium rosenbergii,also known as the giant freshwater prawn,is one of the world’s largest freshwater prawns.It is an important aquaculture species widely cultivated in some Asian nations,such as Bangladesh,China,India,Myanmar,Thailand,and Vietnam.Germ degradation,such as decreasing of disease-resistance and adaptability to environment,has been widely concerned with the rapid growth of M.rosenbergii farming scale.The studies on gonadal development are beneficial to the genetic improvement of M.rosenbergii which is an effective way to prevent germ degradation.FOXL2 has been identified in some species of mammals,fish,arthropods,and mollusks.Previous studies in mammals and fish suggested that FOXL2 is a transcriptional regulator of developmental processes such as gonad differentiation,development,and maintenance.However,the potential function of FOXL2 in crustaceans is not clearly known.Based on this,this thesis mainly studied the bioinformatics characteristics,expression patterns,and biological functions of MrFOXL2(M.rosenbergii FOXL2),laying a foundation and providing reference for the research on the gonad differentiation and development of M.rosenbergii,the research contents were as follows:1.Cloning and sequence analysis of the MrFOXL2To identify the middle fragment c DNA sequence of MrFOXL2,the ovarian transcriptome of M.rosenbergii was screened with fox12 sequence of Procambarus clarkii as a query using BLAST+.The 3’ end fragment sequence of MrFOXL2 c DNA was obtained by using 3’ RACE.The final MrFOXL2 c DNA sequence was assembled using the middle fragment and the 3’ end fragment sequence.The c DNA sequence of MrFOXL2 is 2524 bp in length,including an open reading frame of 1470 bp.The calculated molecular weight(MW)of the MrFOXL2 protein is 53.571 k Da,with an isoelectric point(PI)of 8.34.MrFOXL2 protein contains two N-glycosylation sites and 56 phosphorylation sites.Predicted secondary and tertiary structures of MrFOXL2 protein contain three α-helices and three β-sheets.The results of identity analysis and multiple sequence alignment suggested that MrFOXL2 exhibited a relatively high identity with crustacean orthologues: P.clarkii(57.98%)and Eriocheir sinensis(58.19%).The identity levels were much lower compared to orthologues from mollusks,Mizuhopecten yessoensis(42.64%),and Rapana venosa(41.80%),and vertebrates,Danio rerio(41.67%),Gallus gallus(41.46%),and Mus musculus(38.46%).Phylogenetic tree analysis suggested that MrFOXL2 clustered with arthropod species and it exhibited the closest evolutionary relationship with FOXL2 orthologues in E.sinensis.Comparative bioinformatics analyses of the MrFOXL2 protein indicate that its function in M.rosenbergii may not be the same as in fish and mollusks.2.Expression patterns of MrFOXL2 in different tissues and at different stages of ovarian developmentTo further study the expression patterns of MrFOXL2,the expression of MrFOXL2 in adult M.rosenbergii stomach,brain,gill,hepatopancreas,ovaries,testis,vas deferens,heart,and muscle tissues were determined by semi-quantitative PCR and quantitative real-time PCR using β-actin and 18 s r RNA as internal controls.The transcripts of MrFOXL2 gene in hepatopancreas,ovaries,testis,and vas deferens were located by in situ hybridization,and the changes in expression of MrFOXL2 at different development stages of ovaries were detected.The results of semi-quantitative PCR and real-time quantitative PCR suggested that MrFOXL2 expression was detected in all examined tissues of adult M.rosenbergii,and it was significantly higher in the testis,vas deferens,and ovaries than in other tissues.The results of in situ hybridization demonstrated that MrFOXL2 transcripts were located in the spermatocytes of testes,the oocytes of ovaries,the secretory epithelial cells of the vas deferens,and the secretory cells of hepatopancreas.MrFOXL2 was transcribed in the ovaries at all developmental phases.The highest expression of MrFOXL2 was in stage Ⅰ,whereas the expression was the lowest in stages 0and Ⅱ.In the present study,MrFOXL2 expression was mainly concentrated in the gonads,and it exhibited sexual dimorphism between the testis and ovaries(higher in testis and lower in ovaries),indicating MrFOXL2 gene may play an important role in the gonad development of M.rosenbergii.3.Preliminary study on the function of MrFOXL2 in gonadal developmentTo further study the function of MrFOXL2 in the gonadal development of M.rosenbergii,dsRNA was designed and prepared to interfere with MrFOXL2.The interference effect of different MrFOXL2 dsRNA injected doses on the expression of MrFOXL2 was investigated both in male and female M.rosenbergii.Using q PCR,changes in expression of gonadal development-related genes were analyzed after RNA interference with MrFOXL2.The histological changes of testis were observed after28-day interference with MrFOXL2.The results suggested that different dsRNA injected doses of dsRNA induced different interference effects on MrFOXL2 expression both in male and female M.rosenbergii.The optimal injection dose is 1μg/g.After injection of dsRNA,the interference effect on the expression of MrFOXL2 lasted more than seven days,and MrFOXL2 expression d ECReased to the lowest on the third day.After MrFOXL2 interference,the expression of VG and ECR was significantly down-regulated and CATL expression significantly increased.The expression of IAG gene decreased significantly at different dsRNA injected doses.Male M.rosenbergii from the 28-day interference treatment group showed a clear arrest of spermatogenesis.In contrast,individuals from the control group showed active spermatogenesis.In conclusion,MrFOXL2 is involved in the spermatogenesis of male M.rosenbergii and transcriptional regulation of VG,ECR,CATL,and IAG,which indicates that MrFOXL2 may be related to the gonadal development of M.rosenbergii.