CRISPR/Cas9-mediated Editing Maize Transcription Factor ZmThx20 And ZmPTF1

Maize is an important crop for food,feed,industrial raw materials,has a very important economic value and social significance.Persistently elevated demand for corn to promote the deepening of the study more than a century maize breeding.CRISPR/Cas9 system as one of the latest genome editing technology,has a simple,efficient,flexible,low-cost advantages,has been widely used in a variety of species genome editing research and made many achievements.To carry out the use of CRISPR/Cas9 system knocked important means other maize genes is expected to create excellent breeding material,but also maize gene function analysis.In this thesis CRISP/Cas9 system Trihelix ZmThx20 transcription factor genes and transcription factors ZmPTF1 maize bHLH domain gene having site-directed editing study,verification CRISPR/Cas9 editing system efficiency and specificity of maize.We obtained using particle bombardment Transgenic maize Transgenic plants tested CRISPR/Cas9 editing system efficiency and genetic stability of these two genes,the preliminary results are as follows:I.Method by bombardment good plant expression vector CRISP/Cas9 system into maize immature embryos,transgenic regenerated plants by plant tissue culture.After the transplant survival planting field,selfing or cross sister harvesting seeds2.The use of herbicides on maize T1 transgenic herbicide glufosinate smear screening,preferably antagonistic plants were then PCR.Transgenic material into the vector VK005-02-G3 were detected 160,wherein the positive material 12,the positive rate of 7.5%.In the transfer vector VK005-02-G3-G27 transgenic material,the detector 120,the positive material 13,the positive rate was 10.3%.Using lateral flow detection strip parts 5 PCR positive material,indicating that the expression of the 5 parts materials bar PAT protein encoded by the gene,transgene positive as determined material.this result is consistent with the PCR results.Positive detection of PCR product materials recovered,subcloned sequencing results showed that the amplified bands of interest in line with expectations.Quantification of the expression levels of the target gene to detect gene-editing ZmThx20 plants,clearly the latter expression was significantly lower than the wild type control.3.The positive transgenic corn material using PCR/RE experimental method of detecting a mutation at the target efficiency,the target gene two base changes were detected.Most cases are bases removed,a small number of base mismatches and insertion.In this experiment the editing efficiency of a target gene by two significantly different sgRNA structure,for ZmThx20 gene,single sgRNA editing efficiency was 11.5%-26.1%,double the efficiency of editing sgRNA only 2.8%-9.4%.The target gene ZmPTFl editing efficiency of 18.2%-22.I1%.4.Plant gene editor morphological observation and detection molecules,gene-editing Zm Thx20 seed plant phenotype is shriveled grain,homozygous mutation resemble the body,scanning electron microscopy can be found in the starch granules rough surface,there are many holes.In summary,CRISPR/Cas9 system in maize genes successfully compiled two goals proved CRISPR/Cas9 system can effectively carry out the corn genome modification in the body and create a suitable material for the ZmThx20 genes and gene function studies Zm PTFl.