Study On CRTSPR/Cas9 Editing And Molecular Mechanism Of Pathogenicity Genes In Ascosphaera Apis

Honeybees are highly socialized insects,which have high global economic values,and ecological importance.Currently,the world's breeding bee colonies are under serious threats and are in huge loss due to nutritional stress,natural habitat loss,mite infestations,loss of genetic diversity,pesticides and dedicated diseases.Chalkbrood,which is a worldwide honeybee fungal disease caused by an entomopathogenic fungus,Ascosphaera apis,and it is also a prominent cause of great losses in the beekeeping industry all over the world,especially in China.Occurrence of honeybee colony losses and consequent economic damages has driven researchers to develop new strategies to control honeybee diseases.Among them,genetic engineering works for molecular investigation of pathogenesis,and possible identification of strategies to control the disease shall take preferred issue of research.Based on this,the CRISPR/Cas9 gene editing was employed to study the functions of four genes of A.apis with the objectives of developing new methods for the prevention and control of chalkbrood disease in honeybees,and enhance honeybee health.The main results are as follow:1.Four A.apis strains were collected from four different geographically located apiary for three provinces of China,and their species-specificity was verified by ITS sequencing.The resistance levels of four different strains to hygromycin B were determined by gradient culture,and the sensitive concentration of four strains to hygomycin B was 25?g m L-1.2.Based on the previous studies,four pathogenicity related genes;Nicotinamide Adenine Dinucleotide binding protein(NAD(P)),Versicolorin reductase(Stc U-2),Sterigmatocytin 8-O-methyl transferase(Omt A),and Super killer protein 3(Ski3)were selected for functional verification.The sg RNA were designed and synthesized according to protospacers and protospacer adjacent motifs(PAMs)of checked target genes sequences.3.Protoplast regeneration conditions were optimized.The optimal conditions for protoplast preparation were as follows: pure spores grown for 24 h,and blended,24-36 h young mycelia,grown in potato infusion dextrose broth(PIDB)and digested in driselase enzyme.4.Optimized the genetic transformation conditions of A.apis.It was confirmed that blended,24-36 h young mycelia,grown in PIDB has yielded better mycelium suitable for highest protoplast isolation using driselase enzyme treatment,yielded about 6.71×107 m L-1 of protoplasts,with >90% regeneration.5.40% PEG4000 dissolved in 1mol/L sorbitol based STC solution is used as a protoplast transformation medium,CRISPR/Cas9 has the highest transformation efficiency.Hundreds of hygromycin B resistant protoplasts were regenerated after transformation for all of the four genes.According to the hygromycin B resistance screening,laser scanning microscopic analysis,PCR testing for specific markers: EGFP and hph on the CRISPR/Cas9 vector and sequencing,fourteen positive transformants of two targeted genes(NAD(P))and(Stc U-2)were obtained respectively,and eight mutants were used for further analysis for each of the two genes.6.Even though transformed protoplasts were regenerating on 25?g m L-1 hygromycin B for Omt A and Ski3 genes,none of them have been transformed when checked by PCR amplification,gel electrophoresis observation,and sequencing result analysis.These results indicated that the fungus showed varying responses in terms of protospacer location and concentration of transformation medium.Accurately transformed A.apis mutants have shown highly reduced and non-functional spore cyst development and loss of sporulation,showing direct connection between sexual development and mycotoxin genes(NAD(P)and Stc U-2)in A.apis.7.The pathogenicity of mutant strain was studied.The results showed that the pathogenic index and morbidity of the mutant were significantly lower than that of the primary strain.In summary,CRISPR/Cas9 gene editing is used for the first time in this study to edit the pathogenicity genes of A.apis and obtained effective mutants,which will provide a reference for further study on the functions of pathogenicity genes of A.apis and consequent effective control of disease.