Generation Of Blunt Snout Bream Without Intermuscular Bones Based On Runx2b Gene Editing

The main fish cultivated in China all have some number of intermuscular bones(IBs),which affect its value of processing and edible at a certain extent.Moreover,IBs may took a risk to people when eating fish.Recently,the characters of IBs have been one of the improved characters that has attracted much attention in the field of fish genetics and breeding research.Our previous study proved that runx2 b is the key gene for the IBs formation,and lack of runx2 b gene could obtain a stable zebrafish mutant without IBs.However,no research has focused on the study of blunted bream without IBs.In this study,we used the CRISPR-Cas9 gene editing technology to construct a mutant line with functional deletion of the runx2 b gene in the economic fish Megalobrama amblycephala,and screened out the F0 generation of blunt snout bream with partial deletion of IBs.After its development to sexual maturity,conduct selfinbreeding to obtain F1 generation.When the F1 generation grew to four months,the individuals of F1 generation were scanned with X-ray in vivo to screen for blunt snout bream without IBs,and the mutation types of blunted bream without IBs were identified using positive clones genotyping techniques.Meanwhile,the bone mineral density of wild type and blunt snout bream without IBs were analyzed by Micro-CT.Subsequently,alizarin red staining and X-ray scanning techniques were used to observe the bone development of wild type and blunt snout bream without IBs.Furthermore,differential expression of bone development related genes between wild type and blunt snout bream without IBs were analyzed by q RT-PCR techniques.Using the national standard methods to detect nutrition composition of muscle tissue in wild type(with IBs)and runx2 b mutant F1 individuals(without IBs).The main findings are as follows:1.Generation of runx2 b mutant F0 of blunt snout bream and phenotype and q RT-PCR analysisStructing the F0 generation of runx2 b mutation blunt snout bream by CRISPRCas9 Gene Editing Technology.Agarose gel electrophoresis was used to detect mutations in F0 generation of blunt snout bream,and Sanger sequencing and sequence alignment were used to further verify whether the runx2 b gene was successfully edited.54(70dpf)runx2b mutant F0 generation were random Ly selected for alizarin red bone staining.The result showed that 6 of them showed the characteristics of partial deletion of IBs,the number of intermuscular spines was 43,7,17,14,7 and 14,and the rates of IBs deletion were 33.86%,5.74%,13.60%,11.48%,5.60% and 11.29%,respectively.F0 generation of blunt snout bream(deletion IBs)with functional deletion of the runx2 b gene were random Ly selected to analyze the expression of bone-related genes.The result of q RT-PCR indicated that,due to the loss of runx2 b,the key genes involved in bone mineralization(entpd5a、entpd5b、spp1、scpp1)showed significantly lower expression level in runx2 b mutants F1 individuals without IBs(P < 0.05)that compared to wild-type.However,the tendon related gene tnmd showed no significant difference between the wild-type and runx2 b mutant F1 individuals without IBs.2.Breeding of mutant F1 generation of blunt snout bream without IBs and phenotype and q RT-PCR analysisIn order to obtain the mutant F1 generation of blunt snout bream without IBs,based on the sexual maturity F0 generation of blunt snout bream runx2 b mutant with more than 30% IBs number reduction,F1 generation of blunt snout bream were obtained by F0 generation mutant self-inbreeding.60(45dpf)runx2b mutant F1 generation individuals of blunt snout bream at 45 dpf were selected for alizarin red bone staining.The results showed that IBs of 27(45%)runx2b mutant F1 generation individuals completely lost,and other bone mineralization were normal,such as vertebrae,ribs,neural arch,haemal arch.When runx2 b mutant F1 individuals grew to four months,runx2 b mutant F1 individuals were scanned using X-ray vivo scanning techniques and a total of 316 individuals totally without IBs were screened out from5000 individuals.Furthermore,Micro-CT result showed that runx2 b mutant F1 individuals did not have IBs.And the experimental results indicated that tissue mineral density(TMD)value of vertebrae,ribs,neural arch,haemal arch had no obvious differences between wild type(with IBs)and runx2 b mutant F1 generation individuals(without IBs)at 120 dpf(P>0.05).Due to the loss of runx2 b,the key genes involved in bone mineralization(alpl、bglap、entpd5a、entpd5b)showed significantly lower expression level in runx2 b mutants F1 individuals(120dpf)without IBs(P<0.05)that compared to wild-type,and the osteogenesis related gene sp7 also showed lower expression level in runx2 b mutant F1 individuals without IBs than wild-type.3.Genotype analysis and muscle nutrient contents analysis of mutant F1 generation of blunt snout bream without IBsIn order to identify the runx2 b mutant type in F1 generation without IBs,35 blunt snout breams(without IBs)were random Ly selected for genotype analysis.The experimental results showed that the number of runx2 b targets 1,2,3 mutation types obtained 3(1 insertion,2 deletion)with insertion 2 bp,deletion 5 bp and 8 bp;13(8insertion,5 deletion)with insertion 4 bp,5 bp,6 bp,8 bp,9 bp,16 bp,17 bp and 25 bp;deletion 2 bp,3 bp,4 bp,5 bp and 8 bp;7(1 insertion,6 deletion)with insertion 1 bp,deletion 1 bp,2 bp,8 bp,10 bp,11 bp and 12 bp;respectively.The mutation efficiency of runx2 b targets 1,2 and 3 was 25.71%,94.28%,and 71.43%,respectively.Direct drying method,automatic Kjeldahl nitrogen machine,automatic fat analyzer,and high temperature ashing method were used to detect runx2 b mutant F1 blunt snout bream individuals(without IBs)nutrition composition of muscle tissue for fish at 120 dpf,respectively.Results showed that the basic nutritional composition of wild type(with IBs)and runx2 b mutant F1 blunt snout bream individuals(without IBs)was similar.There were no significant differences(P>0.05)between wild type(with IBs)and runx2 b mutant F1 individuals(without IBs)in crude protein,crude lipid and crude ash.Moreover,moisture in runx2 b mutant F1 individuals(without IBs)was higher than wild type(with IBs),and there was not significant difference(P>0.05).14 fatty acids were identified in the muscle by Agilent gas chromatograph,including 4 saturated fatty acids and 10 unsaturated fatty acids.Furthermore,most fatty acids showed no significant differences(P>0.05)between wild type(with IBs)and runx2 b mutant F1 individuals(without IBs).A total of 17 amino acids were identified in the muscle by automatic amino acid analyzer,including 10 none-essential amino acids and 7 essential amino acids.Additionally,contents of those amino acids in runx2 b mutants(without IBs)were little higher than that of wild type(with IBs),but no obvious differences were indicated(P>0.05).