Cloning And Expression Of Full Length Cdna Encoding Allatostatin (Ast) In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, belongs to Arthropoda, crustaceans, Decapoda, Palaemonidae. M. nipponense which is widely distributed around the country has many advantages, such as less disease, fast growth, nutritious and so on. M. nipponense is considered as a major freshwater aquaculture species of shrimps and crabs. However, with the shrimp farming scale expands gradually from generation to generation, some phenomena have seriously affected the freshwater shrimp farming efficiency, such as species degradation, small size and early sex maturation. As a result, some researches related to the function and mechanisms of molting and reproductive development of oriental river prawn are very urgent. The insects and crustaceans are both arthropod, they have periodic molting and other similar physical characteristics in common. Allatostatin was discovered in insects firstly, it has important function in adjusting molting and reproduction. Crustacean brain cells secret allatostatin (AST), promoting molting, gonad maturation, egg maturation and regulation of reproduction and other important functions. Therefore, this study uses brain as material, in order to obtain the full-length AST gene cDNA sequence and test the expression level of AST in different tissues in oriental river prawn, in hopes making some foundational information for the functional genes analysis. As while as providing reference of the reproductive development mechanism in oriental river prawn.In this research, fragment cloning method based on the traditional method was applied to obtain full-length AST cDNA sequence of oriental river prawn. In other words, the full-length AST cDNA sequence of oriental river prawn was spliced into several gene fragments for amplication. In this study, a total of four middle cDNA fragments were amplified, obtaining the full-length middle cDNA sequence through joining these four middle cDNA fragments together, then joining with the3’ terminus and5’terminus of cDNA, in order to derive the full-length AST cDNA sequences. This study provides foundational information for further molecular biological experiments. The full-length AST cDNA sequence is2995bp, including242bp of5’ untranslated region (UTR),647bp of the3’UTR,2106bp open reading frame (ORF). The upstream region of poly (A) has a significant polyadenylation signal AATAAA, encoding a701amino acid polypeptide precursor in open reading frame, suggesting with molecular weight of78.7kDa and theoretical isoelectric point of9.15. Amino acid polypeptide precursor includes27amino acid signal peptides and674amino acid mature peptides.35different forms can be transcript and translated from the AST peptides due to the alternatively splicing which have the same Y/FXFGL-amide structure at C-terminus, belongs to typical a type of AST because of the same AST peptide structural features in cockroaches. Through the comparison of AST peptides with the freshwater Procambarus clarkii, Carcinus maenas, Penaeus monodon, Panulirus interruptus, M. rosenbergii, the results showed that there are some conserved amino acids, including Tyr, Phe, Gly, Leu, the conserved peptide is AST-19,26,29, oriental river prawn-specific peptides are the AST-3,8,16,20,24. Over60%of AST protein between some insects and crustaceans were similar and AST evolution in invertebrates is relatively conserved. According to the phylogenetic tree analysis, crustaceans and insects are separatly assigned into one branch which is consistent with taxonomic. Interestingly, M. nipponense has the closest genetic relationship with M. rosenbergii.Quantitative PCR (qRT-PCR) technique analyzes the expression level of AST gene in brain, intestine, liver, heart, testis and ovary of oriental river prawn with p-actin gene as control. According to the analysis, AST mRNA was expressed in all of these tissues. The expression level in turns as follows:liver> intestine>testi> brain> heart> ovary. The existing researches showed that AST plays an important role in molting and reproductive of crustaceans. Besides it can affect the frequency and range of intestine and heart’s contraction. In this research, AST has been detected highest expression in liver, and the expression of other organizations have reached a very significant difference. Then in the intestine and testi also have a higher expression. The brain and heart have a relatively low expression AST was express less in ovary, meanwhile, differences between organization not significant.It is the first time to clone and achieve the full length cDNA encoding AST of oriental river prawn in our research. We analysed the structure of AST cDNA. Besides, use the quantitative real-time PCR (qRT-PCR) technique to detect existence of AST gene in various tissues. This result can provide references in AST gene deeper studies in oriental river prawn. Besides, our study makes an example in molting and reproductive of crustaceans.