Molecular Cloning And Expression Analysis Of Vitellogenin Gene From The Shrimp Fenneropenaeus Merguiensis

Fenneropenaeus merguiensis is one of the most important wild shrimp harvested commercially in China.The shrimp also has a high potential for aquaculture in China.To provide important physiological knowledge of breeding for this shrimp,molecular cloning and expression analysis of vitellogenin gene from the shrimp Fenneropenaeus merguiensis were studied.In this study,FmVg1,FmVg2,FmVg3 and FmVg3 X genes were cloned by transcriptome sequencing technique combined with PCR technology.The results showed that F.merguiensis express at least 4 vitellogenin genes.The full length c DNA of FmVg1,FmVg2 and FmVg3 range from 7758-7764 bp and the encoded protein size range from 2553-2585 AA.FmVg1,FmVg2 and FmVg3 genes were highly conserved and all of them contained 15 exons and 14 introns.Among them,the "FmVg1" class Vg was more conservative than the "FmVg2 and FmVg3" class Vg,and all of them had the highest conserved structure in the N-terminal LPD_N domain.Gene sequence analysis showed that the four Vg genes may evolve from gene duplication and nucleotide mutation of a common ancetor gene.During evolution,the ancestor Vg gene was first evolved into FmVg1 and a gene duplication event occurred to produce the FmVg2 followed by nuceotide mutation.FmVg2 was further duplicated into FmVg2 and FmVg3 genes with a higher mutation rate for FmVg3.FmVg3 duplicated again to form FmVg3 X with its coexistence by shearing the intron.As a gene family,the Vg genes could fullfill the nutritional functions of shrimp.To analyze the expression of FmVg1,FmVg2 and FmVg3,we have used immature female shrimp(GSI<1%)for qualitative/quantitative Real Time PCR technique.The results showed that FmVg1 transcript was mainly detected in the hepatopancreas and ovary,while FmVg2 and FmVg3 transcripts were mainly detected in the hepatopancreas and the express level in the ovary was very low.FmVg1,FmVg2 and FmVg3 were all expressed in the hepatopancreas and ovary of mature female shrimp,and the expression levels of the Vgs in the hepatopancreas of males was low.The expression pattern of FmVg1,FmVg2 and FmVg3 in the hepatopancreas was similar in different stages of female gonad development.In ovarian development stage I-III,with the increase of Gonadosomatic index(GSI),the expression level of Vg increased,the expression of IV and V decreased,but remained at a high level.The expression of FmVg1 in the ovary was the same as that in the hepatopancreas.No obvious change in the expression of FmVg2 and FmVg3 in phase I-IV but the expression level was the highest in V phase.The expression levels of FmVg1,FmVg2 and FmVg3 were similar during the molting period of juvenile females and males.The expression levels were low in the late premolt stages and postmolt stage the,the highest amount of expression in the intermolt,with the pattern AB<C1<C23>D01>D23.FmVg1,FmVg2 and FmVg3 transcripts can also be detected at different developmental stages with low expression rate during embryonic development,nauplii,reduced further in the juvenile stage but maintain a high level.To summarize,a contant and low levels of FmVg1,FmVg2 and FmVg3 expresion is occurred in female and male.The hepatopancreas and ovary were the main synthetic sites of Vg in female,while hepatopancreas is the major synthetic site of Vg in male.During the non reproductive seasons,Vg produced by hepatopancreas is most likely,involved in the growth and development of shrimp.However,during reproduction,Vg was synthesized by hepatopancreas and ovary to meet the nutritional needs.The expression patterns of FmVg1,FmVg2 and FmVg3 were similar in the hepatopancreas at different developmental stages,while the expression trends of FmVg1 and FmVg2/3 in ovary was different.High expression rate of FmVg2 and FmVg3 and therefore vitellogenin(or vitellin)was accumulated in the spawning season.During embryonic development,larval growth and juvenile stage,FmVg1,FmVg2 and FmVg3 are required for morphogeneis,metamorphosis of larvae,molting cycle and reproductive cycle and other processes.