Screening And Identification Of ROS Regulation And Growth Trait-related Genes In Penaeus Vannamei

Penaeus vannamei is one of the most popular aquaculture species in the world.In recent years,it has become the most farming area and highest yield shrimp species in China.However,the shrimp culture is faced with some problems,such as frequent disease outbreak,germplasm degradation,long-term dependence on import of germplasm resources and so on.The key to solve these problems is to understand the molecular mechanism of disease resistance and select new lines with good characters.In this process,the traditional breeding method is very time-consuming and costly.Molecular marker-assisted selection(MAS)came into being,which can improve accuracy and reduce the cost.This technique depends on trait-related genes.Therefore,this study purpose is to screen important trait-related genes and to deepen the molecular mechanism understanding about disease resistance and growth traits.It lays a good foundation for MAS technology.The main research is as follows:1.The P.vannamei hepatopancreas cDNA expression library was constructed by Gateway technique.The constructed cDNA was mainly distributed from 500 bp to 5000bp which was consistent with previous reported genome annotated genes and indicated that this cDNA library was integrity and less disruption.The entry clone library capacity was about 9.63×10~6 CFU,which was evaluated by dilution plating,and the mammalian expression library capacity was about 1.79×10~6 CFU.It has been reported that P.vannamei contained 25596 annotated protein-coding genes,thus our expression library has a high coverage rate.2.A novel FACS-based screening method was developed for shrimp ROS regulating gene screening.293T cells was used as bioreactor and the green fluorescent protein(roGFP)was used as redox biosensor.The fluorescence intensity of 293T cells can reflect the intracellular ROS level change.Firstly,the roGFP plasmid was transfected into 293T cells.293T cells with relatively stable expressing roGFP were obtained after 5-6 passages of sorting.Then,the P.vannamei hepatopancreas cDNA eukaryotic expression library was transfected into 293T cells expressing roGFP.We collected ROS-low cells with a cell sorter and reverse transcribed shrimp genes m RNA to cDNA from sorted trace cells.After that,the cDNA was cloned into T vector and sequenced.Totally 85 non-overlapping shrimp genes were identified.The candidate genes were selected and constructed into p IRES2-Ds Red-express vector.After transfection of 293T cells,the changes of ROS level were detected to verify the candidate genes.The results showed that Lyase C29B12.13-like and macrophage mannose receptor 1-like genes could stimulate ROS production.Through this method,we screened the potential ROS regulating genes from P.vannamei hepatopancreas cDNA eukaryotic expression library.3.P.vannamei growth related genes were screened via a modified method.The fluorescent probe can be replaced by 2-NBDG,which can be used as a fluorescent indicator to determine the rate of glucose uptake.Six candidate genes were preliminarily screened by checking the correlation between gene expression and individual weight within one batch.It was confirmed that ATP synthase subunit e and apoptosis inhibitor protein genes were related with the growth rate.And then,we further test these two candidate genes in various lineage and confirmed that ATP synthase subunit e could be a shrimp growth-associated breeding marker.In conclusion,this study developed a new method for screening functional genes from the P.vannamei cDNA eukaryotic expression library.This method is featured with its high coverage,low cost and wide application range.Some ROS regulating genes and growth-associated genes have been screened.It laid a good foundation for further revealing the regulatory mechanism of ROS in P.vannamei,and provided new growth-associated genes for MAS.