The Study Of RNAI Technology And Its Application In Function Study Of Molt-Inhibiting Hormone Gene (MIH) In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, subordinated to Arthropoda, crustaceans, Decapoda, Palaemonidae, Macrobrachium, is an important shrimp species in fresh water aquaculture. In recent years, with the development of the genetic breeding work, a lot of sexual and reproductive related genes had been cloned and analysised of gene expression. But the research on the function of these genes still lack effective means. RNA interference (RNAi) technology is an important mean of gene function research.It had been used in the study of gene function in a variety of crustaceans. However, there were few reports of Macrobrachium nipponense. If the RNAi method can be established in oriental river prawn, it will open up a new path for the study of gene function. Therefore, in this study I selected the transformer-2(tra-2) gene, which has been cloned in our laboratory, for the establishment of RNAi technology. And then, the molt-inhibiting hormone (MIH) was cloned and analysised of spatial-temporal expression. The RNAi method was used in the gene function study of MIH.Mntra-2 was used to study the best injection site, injection dose, interference regularity of RNAi in Macrobrachium nipponense. By comparing the injected site of muscle、heart、 pericardial cavity and the base of eyetalk, we chose the pericardial cavity as suitable injection site of oriental river prawn. Then we performed the time-dependent experiment and the RNAi regular of different doses (0.4 μg dsRNA/g body wt,4 μg/g and 12 μg/g). The qRT-PCR technique was used to detect the tra-2 mRNA expression level. Time dependent experiment showed that the effect of RNAi may change as time changed. The dose of 0.4 μg/g had no interference effect. The dose of 4 μg/g or 12 μg/g had significant interference effect, the tra-2 mRNA expression level decreased by about 80%, and both were presented to recover after falling. The dose of 4 μg/g knocked tra-2 gene expression level down to the lowest level on the 7th day after injection, and the expression level recovered to normal as the control group on the 14th. The dose of 12μg/g make the lowest point advanced to the 5th day, and the interference effect lasted longer:the tra-2 gene expression only recovered 60% compared to the control group on the 14th.The full-length cDNA sequence of MIH gene was firstly cloned of oriental river prawn using rapid-amplification of cDNA ends (RACE) technique. The full length cDNA sequence of MIH gene was 925 bp, containing a 360 bp open reading frame (ORF), encoding 119 amino acids polypeptide precursor, including signal peptides containing 41 amino acids and mature peptides containing 78 amino acids. The comparison of MIH peptides with the Macrobrachium rosenbergii and Cancer pagurus revealed that the amino acid similarity was over 95%. According to the analysis of the Phylogenetic Tree, the genetic distance between M. nipponense and freshwater crabs was short and assigned into the same branch with Macrobrachium. The Quantitative real-time PCR (qRT-PCR) was applied to detect the spatial-temporal expression pattern of MnMIH for the first time. According to the analysis results, the mRNA expression level of MIH was high in Cleavage stage(CS),then fell down.With the development of eyespots in Zoea stage(ZS), the MIH mRNA expression level increased. During the larver it fell to the minimum, and change regularity accompany with the molting cycle in post larver. According to the analysis results, MIH gene has the highest expression level in eyetalk, while lowest in brain. The expression levels in turns as follow:eyestalk> abdominal ganglion>gill>ovary>brain, but not in the heart, muscle, testis and hepatopancreas. During a molting cycle, MIH mRNA gene expression level decreased to the lowest before molting, rising rapidly after molting and maintain high expression level during the intermolt stage.The MIH gene function was researched according to the established RNAi technology. The dose of 4μg dsRNA/g body wt was used in the RNAi experiment, and injected from pericardial cavity. Then the eyetalk of the interfered oriental river prawn was collected for the latter detection. The results showed that, the varying pattern of RNAi group was similar to the tra-2 gene, MIH expression level was the lowest on the 6th to 8th, reduced 92%(P<0.05)compared with the control group. The effect was more significant than tra-2. The M. nipponense at the same development stage was divided into four groups for the continuous RNAi experiment:RNAi group and the control group of male, RNAi group and the control group of female. The injection dose and site were the same as before, but a supplementary injection every 7 days for each animal was needed and lasted for 6 weeks. Results showed that RNAi for MIH gene can significantly increase the molting frequency of male shrimp and female shrimp, the molting frequency of male and female in RNAi groups were 17 and 12 times respectively.While there were only 2 and 5 times in the control groups. The weights of male RNAi group increased 0.26g during the test (P<0.05), however, the other three groups were 0.06g,0.08g and 0.04g respectively, it was not significant increased(P>0.05). It suggested that the MIH gene had the function of regulating the molting, and would inhibit the molt of M. nipponense. Moreover, the weight growth of male shrimp had been influenced.In this study, RNAi technology was systematically studied in oriental river prawn for the first time, and the interference regularity was researched. It was the first time to clone the full length cDNA sequence and analyse the structure of MIH gene of oriental river prawn. Besides, qRT-PCR technique was used to detect the expression level of MIH gene in various tissues, different stages and a molting cycle. Meanwhile, the RNAi technology was applied in the study of MIH gene function for the first time, it proved that the function of MnMIH was to inhibit molting of Macrobrachium nipponense. This study provied basic data for the research of molting mechanism in crustacean. Meanwhile, it provide an effective method for the function study of other genes in Macrobrachium nipponense.