Functional Analysis Of Ovarian Development Related Genes In Macrobrachium Rosenbergii

Macrobrachium rosenbergii is a worldwide prawn breed with high production value,which is famous of its outstanding producing characteristics（e.g.,wide-range diet,short breeding cycle,high nutrition and strong disease resistance）.The volume of the M.rosenbergii industry in China has been growing into the biggest volume since M.rosenbergii was introduced into China in 1976.The growth of the M.rosenbergii industry,however,is considerably restricted by many producing problems,e.g.,“Iron Shrimp”.“Iron Shrimp”refers to an abnormal status that the shrimp cannot grow bigger while shows precocious puberty.Ovarian maturation is the key indicator for female to determine sexual maturation.Thus,it is important to study the mechanisms and candidate genes related to ovarian development for controlling the sexual prematurity artificially and avoiding the occurrence of“Iron Shrimp”.In the study,the transcriptome sequencing is conducted for ovarian tissues sampled from the ovarian in four sequential stages.The differentially expressed genes are screened compared among samples（each pair of four samples）,and performed GO function classification and KEGG pathway annotation.The SSR region is identified,and applied in the association analysis with body weight data.The full-length cDNA of five genes related to ovarian development is cloned.The expression of two genes is silenced by RNA interference,respectively.The main results are shown below.1)In total,221,580,604 clean readsand 95,379 Unigenes are obtained.6,605differentially expressed genes are detected and included 2410 up-regulated and 4,195down-regulated genes.6,422 Unigenes are mainly categorized by GO analysis as,molecular function,cellular component and other biological process.The differently expressed genes are chiefly demonstrated the relations with pathways like carbohydrate transport,amino acid synthesis,enzyme activity and the composition of the cell membrane.KEGG pathway analysis shows 8,423 Unigenes are annotated and linked to 184 pathways（7 pathways are associated with ovarian development）.Differentially expressed gene analysis enriches in pathways,say,Drug metabolism-cytochrome P450,GABAergic synapse,Glycosphingolipid biosynthesis-globo series and Nitrogen metabolism.In addition,SSR and SNP loci are identified.The above results help to understand the ovary development mechanism in M.rosenbergii.2)18,592 SSR loci are identified from the transcriptome sequencing data.63 SSR loci are randomly picked for genotyping.31 loci among them possess the polymorphism.19 loci（out of 31 polymorphic loci）are used for analyzing the population genetic structure of 119full-sib individuals（FS）and 199 wildtype Bangladesh F3 individuals（MJL）.The association analysis is conducted for body weight traits.40 alleles are detected in FS,with an average polymorphism information content of 0.3076.A total of 65 alleles are detected in MJL,with an average polymorphism information content of 0.3882.Both two populations present moderate genetic polymorphism.There is no correlation between 19 SSR loci and body weight trait in FS（P>0.05）,but 4 SSR loci are significantly correlated with body weight in MJL（P<0.05）.3)The full-length cDNA of genes Cyclin A,Cyclin B2,Cyclin E,Cyclin G,Cyclin H are cloned with PCR and 5’-and 3’-RACE.Their full-length cDNAs are 2,140bp,1,642bp,1,635bp,3,427bp and 1,828bp respectively,encode 451,468,421,1,081 and 332 amino acids,respectively.The predicted proteins of genes Cyclin A,Cyclin B2,Cyclin G,Cyclin H contain several glycosylation sites and phosphorylation sites,while the CYCLIN E protein only involves glycosylation sites.The CYCLIN G protein includes S＿TKc domain and a PTEN＿C2 domain,and all other proteins includes CYCLIN domain（a characteristic domain of the CYCLIN family members）.Both proteins don’t have signal peptide and transmembrane helical structure.Phylogenetic analysis demonstrates the structure of the five genes has closer relationship with proteins in crustaceans.4)The gene expression of above five genes can be detected in ten tested tissues（blood,eye stalk,muscle,gill,ovary,testis,hepatopancreas,intestine,heart and stomach）by RT-PCR.The expression level of Cyclin A,Cyclin B2 and Cyclin E is the highest in ovary,followed by them in hepatopancreas.The expression of Cyclin G is the highest in muscle,followed by hepatopancreas.The expression level of Cyclin H is the highest in hepatopancreas,followed by that in ovary.The expression trend is composed for each gene with the expression levels in seven time-nodes(on the 2_nd,4_th,6_th,8_th,10_th,12_th,14_th day of larval breeding).The expression levels decline after turning over the pool（on day 8 of the incubation,the culture was continued in a new environment）.Further,the expression of the five genes is measured at yolk occurs early phase（phase I）,primary yolk occur（phase II）,secondary yolk occurrence period（phase III）,the mature stage（phase IV）,brooding period（phase V）.The five genes express lowest at phase I and highest at phase V（gene Cyclin E expresses highest at phase IV）.These results provide evident for the five genes as the candidate genes with ovarian development.5)The expression of genes Cyclin A and Cyclin B2 is silenced by RNA interference for examining the impact of lacking of the two genes on the ovarian development.The expression of Cyclin A and Cyclin B2 in ovaries decreases significantly after siRNA and dsRNA injections in vitro,respectively.There is no significant difference of the expression level of Cyclin A between the injection group and control 2 hours after injection（P>0.05）.The expression level of the injection group is significantly lower than it in control 4 hours and 6hours after siRNA injection（P<0.01）.2,4 and 6 hours after injecting Cyclin A siRNA,the expression of Cyclin A in the injection group declines by 4.6%,14.6%and 34.6%,respectively,compared to control.In contrast,2,4 and 6 hours after siRNA injection,the expression level of Cyclin B2 in the injection group is significantly lower than it in control（P<0.01）,which decreases by 33.5%,35.4%,and 37.5%respectively compared to control.Notably,injecting dsRNA restrains the expression of genes Cyclin A and Cyclin B2 and affects the ovarian development.Injecting Cyclin A dsRNA postpones the ovarian maturity and Injecting Cyclin B2 dsRNA postpones ovarian development compared to control.