Genetic engineering in probiotics for activation of the interfering RNA (RNAi) system in shrimp: A potential application in the control of viral diseases
The infectious diseases, major viral ethology, has been the main challenge in the shrimp production. The activation of the RNAi (interference RNA) mechanism can promote the control of the viral replication, such as (WSSV viruses White Spot Syndrome Virus) and IMNV (Infectious Myonecrosis Virus). Os trabalhos descritos nessa Tese estão relacionados com o método de produção e entrega de dsRNA (RNA dupla fita) para o camarão branco do Litopenaeus vannamei, com o intuito de ativar o mecanismo do RNAi. The studies described in this thesis are related to the production and delivery of dsRNA (double-stranded RNA) for Litopenaeus vannamei, in order to activate the mechanism of RNAi. On the possibility to produce dsRNA using bacteria was made genomic sequencing and genetic manipulation in native Bacillus strains, as well-made genomics editions in B. subtilis using the CRISPR/Cas9 technology. In the chapter I was sequenced the genome of the two native B. cereus strains isolated from the gastrointestinal tract of crab Ucides sp. from the Pacoti River in Fortaleza - CE. These molecular findings can improve information of this strain and give support for genetic manipulation. In the chapter II, a native B. cereus strain was manipulated to fluorescence production (GFPmut1). In addition, it was delivered in feed and evaluated the colonization in the intestinal tract of the L. vannamei. In the chapter III, plasmids were developed for genomic editing of B. subtilis using the CRISPR/Cas9. In the first moment, rnc gene was deleted and, in the second step, genes coding for GFPmut1, dsRNA against WSSV and IMNV viruses, and unrelated dsRNA were integrated into B. subtilis genome. At the end, two B. subtilis strains (JJBs5 and JJBs8) were produced, both of them free of the antibiotic resistance that could be tested in shrimp bioassays with viral challenge. In conclusion, the findings presented in this work could represent new perspectives to mitigate the problems with shrimp disease in commercial farms.