Screening And Functional Analysis Of Genes Related To Astaxanthin Enrichment In The Ridgetail White Prawn Exopalaemon Carinicauda

Carotenoids are tetraterpene pigments which exist in microorganisms,plants and animals,which include lycopene,β-carotene,astaxanthin,lutein and so on.They are important nutrients for human beings,acting as antioxidants,visual pigments,and even with the essential effects on immune defense.Among these carotenoids,astaxanthin shows the strongest antioxidant capacity with 6,000 times higher than vitamin C,which is known as the "king of antioxidants".It is widely found in crustaceans such as shrimps and crabs,however,astaxanthin cannot be synthesized de novo in animals,which are usually accumulated from diets or modified through metabolic reactions.There are limited studies on the absorption,transport,metabolism and utilization of astaxanthin in animals.In recent years,we found a mutant of the ridgetail white prawn Exopalaemon carinicauda,which have been reared and identified as a new variety“Kesuhong No.1” with high level of astaxanthin.The concentration of total astaxanthin in the orange-red body variant prawns is 10 folds higher than that in the wild type prawns.Therefore,revealing the molecular mechanism of astaxanthin accumulation in the ridgetail white prawn is of great significance for understanding the metabolic process of astaxanthin in animals and improving the quality traits of crustaceans.In this research,the genetic analysis of the astaxanthin enrichment trait of the new variety "Kesuhong No.1" was carried out.The differentially expressed genes and pathways between the new variety and the wild type prawns were identified based on the comparative transcriptome analysis.Additionally,characterization and function analysis of the carotene oxygenase,which contributed to the metabolic processes of carotenoids,were also performed.Those SNPs associated with astaxanthin enrichment trait,which showing the significant genotype differences between the wild type prawns and the orange-red variant,were mined by BSR-Seq.And the trait determining region related to astaxanthin enrichment was analyzed on the basis of the correlation analysis and genetic linkage analysis.The identification and function analysis of candidate gene underlining the astaxanthin enrichment in E.carinicauda was also conducted.These results provide important information for understanding the molecular mechanism of astaxanthin accumulation in E.carinicauda,and essential reference for the breeding strategies in crustaceans.The main research progresses were as follows.1.Comparative transcriptome analysis in the mutant and wild type prawns.To reveal the potential molecular mechanism of astaxanthin enrichment in E.carinicauda,transcriptome analysis was performed between the mutant and the wild type prawns.A total of 78224 unigenes were obtained and 1863 were identified as DEGs,in which 902 unigenes showed higher expression levels,while 961 unigenes presented lower expression levels in the mutant.Based on the GO analysis and KEGG analysis,“Lysosome” was the most significantly enriched pathway,and most pathways in Metabolism were down-regulated in the mutant.The results of q PCR detection also showed that genes related to binding and transport,including crustacyanin,Apolipoprotein D(Apo D),carboxylesterase and cuticle proteins,showed significantly differential expression between the two types of prawns.These data indicated that the biological processes related to astaxanthin transport,metabolism and binding showed significant expression differences between the mutant and the wild type prawns,and these differences may help us find out the mechanism of astaxanthin enrichment in E.carinicauda.2.Identification and functional analysis of enzymes related to carotenoid cleavage in E.carinicauda.Carotene oxygenases can contribute to the degradation of carotenoids,and some researches have shown that they have important effects on carotenoid accumulation in animals.In our study,nine homologous genes were identified in the transcriptome database of ridgetail white prawn,including seven Ec BCO-like genes and two carotenoid isomerooxygenase-like genes.Phylogenetic analysis showed that these two carotenoid isomerooxygenase-like genes completely separated from Ec BCOlike genes,and Ec BCO-like genes except Ec BCO-like1 were clustered together.Additionally,tissue distribution analysis showed that Ec BCO-like genes were highly expressed in tissues like hepatopancreas or stomach,while carotenoid isomerooxygenase-like genes were highly expressed in the eyestalk.This indicated that these two types of genes might play different roles.We then conducted the RNA interference on Ec BCO-like1,Ec BCO-like3 and Ec BCO-like6 for functional research,and results showed that the mortality rate of these prawns increased rapidly after knockdown of Ec BCO-like1.This indicated that Ec BCO-like1 might be of importance in fundamental metabolism process.No phenotype change was observed after Ec BCOlike3 knockdown,while prawns injected with ds Ec BCO-like6 showed color changes in their hepatopancreas and the content of carotenoid in their hepatopancreas was much higher than that in the control prawns.This result suggested that Ec BCO-like6 may play an important role in carotenoid metabolism.3.Mapping and genetic analysis of genes related to astaxanthin enrichment in E.carinicauda.Polymorphic SNPs were identified by using the BSR-Seq data and verified in the mapping population F1,F2.A total of 559 SNP markers associated with astaxanthin enrichment were identified,and they were distributed on 347 genes.Then three genes including Unigene0053862,Unigene0016297 and Unigene0027594 were found tightly linked to the trait and located on the same scaffold.In addition,the expression analysis of candidate genes showed that Unigene0053862 and Unigene0016297 were with no difference in the expression level between the new variety and the wild type prawns,while Unigene0027594 was differentially expressed in these two populations.Therefore,Unigene0027594 was identified as the candidate key gene that was tightly linked to the mutant.4.Identification and functional analysis of candidate gene related to astaxanthin enrichment in E.carinicauda.There was only one polymorphic SNP in the c DNA sequence of Unigene0027594 between the new variety and wild type prawns,and the SNP was located in the non-coding region.Expression analysis showed that this gene was expressed in all tissues of wild type prawns,but the expression level in the new variety was extremely low.Through analyzing its expression at different developmental stages,we found that Unigene0027594 was highly expressed in the wild type prawns since the nauplius stage.However,the expression level of Unigene0027594 was always significantly lower than that in the wild type prawns at different developmental stages.In addition,the expression of GFP-Unigene0027594 fusion protein revealed its localization on the cell membrane.After knockdown of Unigene0027594 in the wild type prawns,the expression level of this gene was lower but there was no apparent phenotypic change.In order to understand the reasons for its differential expression in the new variety and the wild type prawns,the sequence of the promoter region of Unigene0027594 was analyzed,and the result showed that a fragment about 5 kb was inserted at the position about 1 kb before the initiation codon in the new variety.In addition,the promoter activity was determined by dual-luciferase reporter assay,and the analysis showed that the promoter of Unigene0027594 was located behind the inserted fragment,and the insertion of the fragment will result in a substantial decrease in promoter activity.These results suggested that the inserted fragment in the promoter region of Unigene0027594 may be the key factor of astaxanthin enrichment in E.carinicauda.