Construction Of CRISPR/Cas9 Targeted Editing Vector For EfPDS Gene And Establishment Of Genetic Transformation System Of Echinochloa Frumentacea

Echinochloa frumentacea(Roxb.)Link is a kind of high-quality forage and crops,and is an important forage and economic crop for cultivation in saline-alkali land.However,the genetic structure of Echinochloa frumentacea(Roxb.)Link is complex and the genome is huge,so there are few studies on Echinochloafrumentacea(Roxb.)Link in China,and the progress in crop genetic breeding and crop improvement is lagging behind.Gene editing has a short breeding cycle,can precisely locate and edit target genes,effectively remove false positives,and improve breeding efficiency.It is widely used in molecular breeding.The establishment of a genetic transformation system is a prerequisite for successful gene editing.At present,there have been reports on the genetic transformation of forage grass,but the research progress of it is still at the determination of physiological indicators.Therefore,the use of gene editing to carry out molecular research on Echinochloa frumentacea(Roxb.)Link has been forced by the situation.The Phytoene desaturase(PDS)gene is the reference gene for verifying the success of plant gene editing.Phenotypic changes can be seen during the budding period of plants,which greatly shortens the time for system establishment,and the construction of The vector provides the basis for the subsequent knockout of other genes.Based on the above reasons,this experiment took Echinochloa frumentacea(Roxb.)Link as the research object,established its complete regeneration system,cloned the partial gene sequence of EfPDS,constructed CRISPR/Cas9 targeted editing vector,and used Agrobacterium tumefaciensmediated technology to initially explore its genetic transformation system,and obtained its gene editing and regenerated plants,laying a molecular basis for its quality genetic improvement.The research results are as follows:1.Establishment of the regeneration system of Echinochloa frumentacea(Roxb.)LinkTaking N003,N004 and Ningji No.1 as the research objects,mature seeds and sterile seedlings were used as explants to induce callus.Through repeated experiments,it was found that the main factors affecting the establishment of their regeneration system were:Explant species,basal medium and hormone types and concentrations.Mature seeds were used as callus-inducing explants.After adding 4.0 mg/L 2,4-D to the final 1/2 MS,callus with good growth could be induced in N003 and Ningji No.1.The average callus recovery rate was 89.71%,and the callus bad obvious granular shape,no water stains,light yellow color and good condition.Adding 2 mg/L KT and 0.5 mg/L 6BA to MS basal medium had the best differentiation effect,and regenerated seedlings could be differentiated in 7-10 days.The rooting ability of regenerated seedlings was best in 1/2 MS medium without any hormones.Finally,the differentiated and regenerated complete plants were hardened and transplanted into a soil:vermiculite(3:1)matrix,and the transplanting survival rate was 100%.When sterile seedlings were used as explants,the induction rate was lower than 20%after repeated conditional screening,and the callus was severely water-stained,which was not conducive to further subculture.Therefore,it was determined that a complete regeneration system was constructed using mature seeds as explants.2.Cloning of Echinochloa frumentacea(Roxb.)Link EfPDS gene and construction of targeted editing vectorIn this study,the CRISPR/Cas9 gene editing technology was used,and the EfPDS was used as the target gene.First,a part of the CDS sequence of the EfPDS gene was amplified by the homologous cloning technology.The fragment was 1068 bp in length.A double target site was designed at the second exon.CmYLCV-pBUE411 was used as the vector backbone,the CmYLCV virus promoter was used to bind tRNA to express sgRNA,the Bsal restriction site was added to the target site,and the maize UBI promoter was used to express Cas9,and the pBUE411 vector was used as the vector The template,after codon optimization of the Cas9 sequence,successfully constructed its EfPDS gene double-target gene editing vector.3.Establishment and molecular identification of Echinochloa frumentacea(Roxb.)Link genetic transformation systemThe successfully constructed CRISPR/Cas9 gene editing vector recombinant plasmid was transformed into Agrobacterium GV3101,the colony was identified as a positive single clone and the induced callus was infected.Repeated experiments confirmed that the optimal transformation conditions mediated by Agrobacterium were as follows.The OD value of bacterial liquid is between 0.6-0.7;the infection time of Agrobacterium is 11-15 min;the suitable co-cultivation time is 2 d.The most suitable subculture medium was 1/2 MS,the induction medium was MS+2.0 mg/L KT+0.5 mg/L 6-BA+500 uL cef,the rooting medium was 1/2 MS,anti-Sexual screening medium is MS+4 mg/L 2,4-D+0.5 mg/L 6-BA+1g/L L-proline+screening agent glufosinate 1 mg/L+cef 300 L(pH7.0),Finally,the genetically transformed plants gene editing were obtained and subjected to PCR molecular identification.The vector plasmid and wild plant DNA were used as positive and negative controls,respectively,and CmYLCV-SEQ and CmYLCV-R were used as primers to amplify the fragment size of 531 bp is the positive plant and the verification result is correct.