| In recent years,gene editing technologies,such as ZFN(Zinc-finger nucleases),TALEN(Transcription activator-like effector nucleases)and CRISPR/Cas9 system(Clustered regularly inter spaced short palindromic repeats/CRISPR-associated proteins),have developed rapidly.More and more attention has been paid to the genetic manipulations by knocking out methods to study gene function through analysis of the biological phenotype and expression of related genes.Among them,the CRISPR/Cas9 technology has been widely used due to its obvious advantages including flexibility,simplicity,and high efficiency based on high-throughput operation,and has become a research hotspot.However,CRISPR/Cas9 method is less studied in marine fish although it has been more used in the model fish and freshwater fish.Olive flounder Paralichthys olivaceus is an important maricultivated fish species in China,Korea,and Japan.The growth and sex traits are important for its culture and genetic breeding.So,a propriate CRISPR/Cas9 technique should be established in the flounder,which can help clarify its gene function and the mechanism of related economic traits,and then guide the genetic breeding.In this study,the technologies for the exogenous substances transferred into the flounder fertilized eggs based on microinjection and electroporation methods and the CRISPR/Cas9 system have been primarily established.The PGC migration factors including atypical chemokine receptor 3b(cxcr7b)and staufen double-stranded RNA binding protein 2(stau2)genes in the flounder were cloned,and then studies on their expression patterns and editing were performed.The effects of these two genes as well as stromal cell-derived factor 1(sdf1)and chemokine(C-X-C motif)receptor 4B(cxcr4b)on PGC migration in the flounder were analyzed with in situ hybridization.At the same time,the dynein axonemal light intermediate chain1(dnali1)gene was cloned in the flounder and highly expressed in the testis.Its differential regulation analysis was also studied.These results will provide a basis for the construction and application of gene editing platform in marine fish,as well as a platform for their gene function study.The main results are shown as follows.Firstly,the technologies for the exogenous substances transferred into the flounder fertilized eggs based on microinjection and electroporation methods have been primarily established.Rhodamine and plasmid were injected into the flounder fertilized eggs at one to four cell stages.The results of express activity showed that the exogenous substance could be effectively introduced into the fertilized eggs by microinjection.Electrorotation method with Electroporator NEPA21 was performed to test the conditions and the results showed that the proper electroporation condition was 3 pulses for 1 millisecond(ms),50 ms interval,at 25 V with high survival rate.Based on the established methods of microinjection and electroporation,gondal somatic cell derived factor(gsdf)and myomaker genes were used for knocking out.Insertion and deletion mutations of gsdf and myomaker were both detected by using these two techniques,and the mutation rate of microinjection was higher than that of electroporation.In order to raise the mutation efficiency of the microinjection technique,concentrations of Cas9 m RNA and g RNA and number of g RNA site were tested.The results indicated that the editing efficiency at low concentration condition with one gRNA site(14.3-45 %)was lower than those at high concentration condition with two g RNA sites(60-80 %),and the high concentration of the injection conditions is more suitable for gene editing in the flounder.Secondly,the flounder cxcr7b and stau2 were cloned,and their expression patterns of them and sdf1 and cxcr4 b in the embryonic development and adult tissues were analyzed.The RT-PCR results showed that sdf1 and stau2 were expressed from the first one cell stage,while cxcr4 b and cxcr7 b were expressed from the blastocyst stage.In the adult flounder,sdf1,cxcr4 b,cxcr7b,and stau2 were expressed in many tissues including gonads,kidney,eye,heart,and liver.The expression of sdf1 in the testis was higher than that in the ovary.The expression of cxcr4 b,cxcr7b,and stau2 in the ovary was higher than that in the testis.Their gene editing experiments through the above CRISPR/Cas9 method were carried out,and all of them obtained first hatched larvae with effective mutation.The mutation rates of sdf1 and cxcr4 b were lower(20-30 %)than those of cxcr7 b and stau2(70-80 %).Furtherly,dynein axonemal light intermediate chain 1(dnali1)gene showing higher expression in the testis was screened according to the transcriptome data of the flounder gonads.The coding region of the gene contains 771 bp,encoding 256 amino acids.There were two alternatively spliced variants of different sizes in the first exon in 5’UTR,and the larger one was only found in the testis but almost not in the ovary.Comparison of the expected transcription factor binding sites of these two variants sequences presented that one SOX5 and one SOX10 binding sites were lost in the shorter one.In adult fish,dnali1 was mainly expressed in the testis,while less in the ovary.Two repetitive fragments,922 bp fragments,were detected in the genome and gene sequences of dnali1,which stretched across its ORF region and 3’UTR region.The 3’UTR regulation activity of the flounder dnali1 was analyzed with transient expression analysis in zebrafish.It was suggested that the 922 bp repeated sequence had dual function,which could control specific translation and maintain RNA stability.This is the first report of dnali1 gene in marine fish,and two spliced variants were also found for the first time.It will provide evidence for the analysis of spermatogenesis and ciliary development in the flounder. |
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