Spatio-temporal Expression Pattern And Functional Validation Of The Csf1r Gene In Monopterus Albus

Monopterus albus,commonly known as eel,is one of the most important economic fish in China.It was found to be sequential hermaphroditic,of which molecular mechanism has been extensively investigated.Molecular mechanism of their different body color patterns was rarely studied.Many studies had showed that different strains had significant differences at growth rate,fecundity,and muscle nutrient composition.Based on the genome-wide association analysis of different strains of Monopterus albus,the result showed that csf1 r gene may be related to the formation of different body colors in Monopterus albus.Investigations in zebrafish had revealed that the csf1 r gene was crucial for maintaining the normal development of xanthophore and played a vital role in the formation of black stripes.In this study,spatio-temporal expression characteristics and function of the csf1 r gene in Monopterus albus were investigated using q PCR and CRISPR/Cas9 gene editing technology.The main results were as follows:1.Cloning and spatio-temporal expression pattern of csf1 r gene in Monopterus albusThe cDNA sequence of Monopterus albus csf1 r gene was cloned from the c DNA of skin by RACEs(Rapid Amplification of c DNA Ends).The deduced amino acid sequence was analyzed.Express levels of Monopterus albus csf1 r gene were detected in different tissues,developmental stages of embryos,and the skin and kidney tissues of Monopterus albus with three kinds of body colors(black-yellow brinded Monopterus albus,smallbrinded and hidden-brinded Monopterus albus).The non-specific immune enzyme activity in the liver of Monopterus albus with three kinds of body colors was determined.The full-length c DNA of csf1 r was 4430 bp in length(Gen Bank accession number:OP589303),with open reading frame of 2937 bp.Csf1 r encodes 978 amino acids including two conserved domains,the immunoglobulin domain and the protein kinase catalytic domain.The highest level of csf1 r expression was detected in spleen and heart,with lower level in kidney,skin and muscle,and the least in the ovary.The expression of Monopterus albus csf1 r was first detected during the embryo stage of lens formation,while pigment granules were present in the embryo torso.The above results suggested that csf1 r gene might be involved in the process of body color formation and play a role in the process of natural immune.This study can lay a foundation for further studies on the molecular mechanism of body color formation and the variety breeding of Monopterus albus.2.Functional validation of csf1 r gene in Monopterus albusTo further explore the function of csf1 r,the powerful CRISPR/Cas9 gene editing technology was employed,and two targets were designed according to the Monopterus albus csf1 r gene.The corresponding g RNAs were prepared,and in-vitro studies showed that target sequences were cut by both sg RNA csf1r-87 and csf1r-513.The two synthesized sg RNAs were mixed with Cas9 protein according to 1 μL mixture containing100 ng of g RNA and 0.2 μL of Cas9 protein(1μg/μL).And then the mixture was injected into the fertilized eggs by microinjection before the two-cell stage.Three samples of Monopterus albus(named M1,M2,and M3)with obvious loss of body pattern were chosen by phenotypic observation.M1 and M3 lost larger areas of pigment and could not form a good stripe pattern than M2.To identify the specific mutations in Monopterus albus mutants,DNA and RNA of the wild-type and three Monopterus albus mutants were extracted,and PCR cloning and Sanger sequencing using specific primers were performed.The results showed that several different mutation types,including base mutations,small fragment deletions,large fragment deletions,and base insertions were present in the mutants,both at the DNA and m RNA levels.Most of these mutations caused gene shift mutations of csf1 r.Micrographs of paraffin sections revealed that the wild-type Monopterus albus had a distinct epidermis layer with a large number of mucus cells in skin.However,the epidermis layer of the mutant Monopterus albus was significantly thinner,with fewer or almost no mucus cells.These findings suggest that the csf1 r gene may play an important role not only in the formation of the stripe pattern but also in the immunity of Monopterus albus.