Yuying Sun

CRISPR/Cas9-Mediated Genome Editing and Mutagenesis of EcChi4 in Exopalaemon carinicauda

The development of the type II clustered regularly interspaced short palindromic repeats (CRISPR) system has resulted in the revolution of genetic engineering, and this technology has been applied in the genome editing of various species. However, there are no reports about target-specific genome editing in shrimp. In this research, we developed a microinjection method for the ridgetail white prawn Exopalaemon carinicauda and successfully applied CRISPR/Cas9 technology to the genome editing of E. carinicauda. Through coinjection of mRNA of Cas9 nuclease and gRNA specialized for E. carinicauda chitinase 4 (EcChi4), shrimps with indel mutations were obtained. Further analysis showed that the mutations could be transmitted to the next generation. This is the first time that site-specific genome editing has been successfully demonstrated in a decapod, and will further contribute to the study of functional genomics in decapods.

Preparation of d-glucosamine by hydrolysis of chitosan with chitosanase and β-d-glucosaminidase

Crude enzymes including chitosanase and β-D-glucosaminidase were obtained by centrifugal separation from the fermentation broth of Microbacterium sp. OU01. Then the crude enzymes are used to hydrolyze chitosan for producing D-glucosamine (GlcN). The effects of temperature, pH, substrate concentration, the ratio of enzyme to chitosan, and hydrolysis time on the productivity of GlcN were discussed. The experimental result showed that the optimal conditions were temperature 50 °C, pH 5.8, substrate concentration 20 mg/mL, the optimum ratio of enzyme to chitosan 1.5 U/60 mg. Under above conditions, chitosan was completely hydrolyzed in 5 h. These results provide a scientific material for the optimization process of enzymatic production of GlcN. What is more, thin layer chromatography and high performance liquid chromatography were used to analyze hydrolytic product, which was proved to be GlcN.

A CRISPR/Cas9-mediated mutation in chitinase changes immune response to bacteria in Exopalaemon carinicauda

Abstract Chitinase, belonging to family 18 glycosyl hydrolase, is a multi‐gene family and it has many functions. Generation of loss‐of‐function mutant targeting an interesting gene is a common way to clarify its function based on reverse genetics. In this study, we first reported the immune defense of a chitinase gene (EcChi4) in Exopalaemon carinicauda using its EcChi4‐deletion mutant. EcChi4 was predominantly expressed in hepatopancreas and was upregulated after challenge with Vibrio parahaemolyticus or Aeromonas hydrophila. After knockout EcChi4 gene using CRISPR/Cas9 tool, the prawns in EcChi4‐deletion group had significant higher mortality than those in wild‐type group when the prawns were challenged with V. parahaemolyticus or A. hydrophila. In conclusion, we first demonstrate the function of a chitinase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis. In the future, CRISPR/Cas9 should be widely applicable as a feasible means for gene editing in E. carinicauda for the study of important biological questions that cannot be easily addressed in other decapods. HighlightsEcChi4 was predominantly expressed in hepatopancreas of E. carinicauda.EcChi4 was upregulated after challenge with V. parahaemolyticus or A. hydrophila.EcChi4‐deleted prawn had higher mortality than wild‐type after bacterial challenge.We first demonstrate the gene function of prawn by performing gene mutagenesis.

Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05

A strain producing chitin deacetylase (CDA) was isolated and identified as Rhodococcus erythropolis by morphological characteristics and 16S rDNA analysis, named as R. erythropolis HG05. By Plackett-Burman and central composite design, CDA production from R. erythropolis HG05 was increased from 58.00 U/mL to 238.89 U/mL. With the crude enzyme from R. erythropolis HG05, the hydrolysate components from colloid chitin were chito-oligosaccharides with polymerization number larger than hexaose.

Enzymatic characterization and functional analysis of EcChi3C from ridgetail white prawn Exopalaemon carinicauda

Chitinase belongs to the glycosyl hydrolases family 18 and plays key role in the development and pathogen resistance of crustaceans. In this study, the enzymatic characterization of chitinase 3C (EcChi3C) of Exopalaemon carinicauda was analyzed. In addition, we analyzed the expression profiles of EcChi3C at different tissues and different molting stages. In the all tested tissues, it was predominantly expressed in hepatopancreas, and then stomach, but poor in other tissues. In all tested molting periods, it was mainly expressed in intermolt and molting stages, but poor in other stages. The results of molting, mortality and the uropod ultrastructure of prawns after being injected with EcChi3C dsRNA were in accordance with those of the control group. In addition, there is no difference for endopodite morphology between the survival and dead individuals in experimental group. After being challenged with bacteria, the expression of EcChi3C was up-regulated significantly at 12 h and followed with a comeback at 96 h. These results suggest that EcChi3C is an important immune related gene but not a necessary gene in the molting process of E. carinicauda.