A Preliminary Study On The Structure And Function Of The Ecdysone Response Genes Of The Pacific White Shrimp Litopenaeus Vannamei And Their Regulation To Wnt Signal Pathway

Molting plays vital important roles in shrimp development,growth and reproduction,and its research has tremendous theoretical and economic values.However,the regulatory network of molting signal pathway in crustacean is incomplete.What and how the ecdysone response genes to regulate downstream genes to achieve molting and growth is still unclear.In this study,we characterized the structures and functions of ecdysone response genes and analysed the relationship between molting signal pathway and Wnt signal pathway based on transcriptome and genome data from the pacific white shrimp,Litopenaeus vannamei.We screened and characterized 20 E early response genes,Lv EcR,Lv RXR,Lv E75,Lv HR3 and Lv FTZ-F1.The sequences analysis showed that the five molting related genes had several splicing forms.In L.vannamei,Lv EcR had eight alternative spliced isoforms;Lv RXR had 4 isoforms;Lv E75 had 6,Lv HR3 and Lv FTZ-F1 has 5 and 4 isoforms,respectively.These isoforms are the results of 3? and/or 5? UTR or extron splicing,or intron conserved.Based on L.vannamei transcriptome data,five 20 E early response genes showed different expression patterns in different development stages,molting stages and adult different tissues.Together with all results showed that the five genes were transcripted in several isoforms in embryo and larvae,while in adult,every gene was transcripted in one or two isoforms.Silencing of Lv E75,Lv HR3,Lv Br-C and Lv FTZ-F1 using double-stranded RNA(ds RNA)caused a significantly decline of the cumulative molting rate and a greatly increase of the cumulative death ra te.The dead or dying shrimp were failed to complete molting in head and/or tail of shrimp.These results indicated that Lv E75,Lv HR3,Lv Br-C and Lv FTZ-F1 in shrimp played important roles in molting process and survival.In order to investigate the relationship between molting and growth in shrimp,we focused on Wnt signal pathway that is critically involved in development and growth.We described 12 Wnt genes representing 12 distinct Wnt gene subfamilies in L.vannamei.Based on homolog annotation and phylogenetic analysis,these 12 Wnt genes named as Lv Wnt1,Lv Wnt2,Lv Wnt4-Lv Wnt11,Lv Wnt16 and Lv Wnt A.All Lv Wnts were single-copy genes.Interestingly,Lv Wnt1 and Lv Wnt6 were adjacent in the same scaffold in the shrimp genome.All the investigated LvWnt genes were initially expressed at the gastrula or limb bud embryo stages,and last to post larvae.Only parts of them were expressed in adult shrimp,as Lv Wnt5 and Lv Wnt A.We used RT-qPCR to assess the expression levels in different development stages consisting with transcriptome data.To further understand the crosstalk between molting and growth,we used qRT-PCR to detect the expression levels of LvWnts,LvZW3 and LvSulf-1 genes in 20E-treated and ecdysone related gene interferenced samples.The results showed that LvWnt genes were significantly regulated by 20 E,while LvEc R,LvRXR and LvE75 genes had little influence on LvWnt genes.In addition,Lv ZW3 and Lv Sulf-1 were different controlled by 20 E and ecdysone response genes.Lv ZW3 was up-regulated in 20 E treated samples;Lv Sulf-1 was down-regulated in Lv EcR interferenced samples;Lv ZW3 was down-regulated significantly and Lv Sulf-1 was up-regulated in Lv RXR interferenced samples;Lv ZW3 and Lv Sulf-1 were up-regulated in Lv FTZ-F1 and Lv Br-C interferenced samples.In conclusion,we hypothesize that the molting and growth of shrimp are controlled by different signal pathways,and there are crosstalk between two pathways through a few genes.