Studies On Reproductive Function Of A-type Allatostatin (AST-A) In The Mud Crab Scylla Paramamosain

Allatostatin(AST)is a class of polyphenic peptide families that inhibit the synthesis of juvenile hormone(JH)by corpus allatum(CA)in insects.According to their structural characteristics,they were classified into three types:A-type AST(AST-A),B-type AST(AST-B)and C-type AST(AST-C).In insects,AST family neuropeptides have a variety of biological functions,among which is to affect the reproductive process considerable.By far,there have been few researches about AST in crustaceans,especially on its reproductive regulation.In order to explore the effect of AST-A on the reproductive process of crustacean,we used qRT-PCR,RACE,in situ hybridization,in vitro culture and RNA interference.We obtain SpAST-A and its receptor SpAST-AR cDNA fragment.Then we examine the tissue distribution and expression in cerebral ganglia at different developmental of SpAST-A gene.We also detect the expression difference of SpAST-AR in ovary at different developmental stages.The expression of SpAST-A and SpAST-AR in the brain ganglia and ovary were analyzed by in situ hybridization.The small peptide was added in vitro and the real-time quantitative PCR was used to detect the expression of the related genes.The expression of related genes was detected by qRT-PCR after interfering withSpAST-AR gene.The main results are as follows:1)SpAST-A neuroendocrine study:The gene fragment of SpAST-A was cloned to be 676 bp,encoding 225 amino acids.According to the conserved structure of SpAST-A,10 small peptides of AST-A could be found from the encoded amino acid sequences.SpAST-A distributed in all the 6 detected tissues(such as ovaries,cerebral ganglia and etc),with the highest transcription levels in the cerebral ganglion.The expression of SpAST-A in the cerebral ganglia at different developmental stages were detected,with the minimum transcription level at previtellogenic stage and the maximum level at early vitellogenic stage.In situ hybridization results showed that SpAST-A was located in the tenth and eleventh cell clusters in the cerebral ganglion.These findings suggested that the brain was an important source of SpAST-A synthesis.2)SpAST-A reproductive autocrine research:The gene fragment of SpAST-AR was cloned to be 704 bp,encoding 234 amino acids,and amino acid sequence analysis showed that there were five transmembrane structures.The homology analysis suggested that it belonged to the G protein coupled receptor family.Both the expression of SpAST-A and SpAST-AR in the ovariy at different developmental stages were detected and their variation tendency were similar,with the lowest expression level at previtellogenic stage and the highest level at late vitellogenic stage.In situ hybridization results showed that SpAST-A and SpAST-AR were both localized in oocytes.Thus SpAST-A probably regulate the ovary development in an autocrine way.The expression of Cyclin B and VgR genes in the ovary were both inhibited by in vitro addition of two small peptides of SpAST-A(AST-A1-4 and AST-A1-5)at low concentration(10-7M and 10-9M),with the most inhibitory effect at the concentration of 10-9 M.The relative expression of SpAST-AR gene was increased by these two small peptides.We hypothesized that,the combination of small peptides of SpAST-A with their receptors could inhibit ovarian development and vitellogenesis.Preliminary interfering SpAST-AR via RNAi technique before addiction of small peptides of SpAST-A could relieve the inhibitory effect on Cyclin B and VgR gene.This result could also prove that the inhibition of SpAST-A on ovarian development and vitellogenesis.In summary,small peptides of SpAST-A may inhibit ovarian development by neuroendocrine and autocrine.The results can provide scientific basis data for crustacean breeding aquaculture technology and have theoretical significance and potential application value.