Gene silencing at the transcription and translation level is a powerful tool for studying gene function and for molecular therapeutics. RNA interference (RNAi) is a key method that enhances scientific knowledge by discovering the function of a wide array of genes and molecular mechanisms. The knowledge obtained from RNAi studies led to novel gene therapies, improved agriculture crops, and new gene intervention technologies for the aquaculture industry. However, a stable silencing and successful introduction of long dsRNA stretches int o cells and oocytes of oviparous animals still needs investigation.
In the current study, a chimeric protein composed of dsRNA binding domain retrieved from M. rosenbergii dsRNA binding protein and an oocyte-specific targeting amino acids sequence (Vg-dsRBD) was cloned, expressed, and purified. The newly developed chimeric protein was found to interact with dsRNA. The Vg-dsRBD bound with dsRNA was characterized by EMSA and TEM. Moreover, the endocytosis capability of dsRBD-Vg bound dsRNA into the oocytes of M. rosenbergii and its capability to silence specific genes was tested by in vitro and in vivo experiments . I ndeed, our in vitro results demonstrate that the novel dsRBD-Vg protein can deliver the bounded dsRNA into M. rosenbergii oocytes . Moreover, the dsRNA introduced to the oocytes by the Vg-dsRBD could silene the eye development PAX6 gene in an in vivo experiment. Such a tool can affect thousands of embryos by administering the complex of the chimeric protein attached to the gene-specific dsRNA into vitellogenic crustacean females . Therefore , the newly developed chimeric protein tool possessing ef?cient gene silencing can be used for silencing crustacean’s aquaculture- relevant genes that may enhance the aquaculture industry.