Optimization Of Cucumber Genetic Transformation System Mediated By Agrobacterium And Creation And Identification Of T-DNA Insertion Mutants

Cucumber(Cucumis sativus L.)is an important horticultural and economic crop in the genus Cucurbitaceae of Cucurbitaceae,with a long history of cultivation.Due to the existence of biotic and abiotic stress in production,it has a great impact on the yield and quality of cultivated cucumbers.Therefore,in order to ensure the yield and quality of cucumbers,new cucumber varieties need to be continuously selected.However,due to factors such as long breeding years,few resistance genes,and low efficiency,the traditional cucumber breeding methods make it difficult to quickly obtain excellent varieties that meet people’s needs.Transgenic technology is an important means of modern molecular breeding,and it plays an important role in analyzing gene function,genetic engineering breeding and genome editing.The Agrobacterium-mediated genetic transformation method is the most widely used and widely used in cucumber transgenic technology,but the transformation efficiency of this method still needs to be further optimized.This study optimized the Agrobacterium-mediated transformation system of cucumber genetic transformation from aspects of Agrobacterium’s own vitality,co-cultivation temperature,Agrobacterium infectious solution concentration,and co-cultivation time.The optimal relevant transformation conditions were determined through data statistics.With the further development of cucumber research at the molecular level,more and more genes with important functions have been located and cloned,and the study of gene functions has become increasingly important.Mutants are closely related to their corresponding mutant genes and can be directly and effectively used for gene function research.However,because of its narrow genetic basis and low natural mutation frequency,the probability of obtaining spontaneous mutants is extremely low.Therefore,artificially creating mutants for research on important functional genes has gradually become a trend.Mutation creation methods include physical mutagenesis,chemical mutagenesis,and insertion mutations.T-DNA insertion mutations in insertion mutations have many advantages such as the ability to study non-phenotypic genes,easy access to insertion mutation sites,and fewer mutation sites.They are suitable for the creation of mutants and gene functions.In this study,the GFP reporter gene was used to optimize the cucumber genetic transformation system,and the creation of cucumber T-DNA insertion mutants was performed.The findings are as follows:1.Optimization of cucumber genetic transformation system mediated by agrobacteriumBased on the Agrobacterium-mediated genetic transformation system of cucumber cotyledonary nodes established in this laboratory,by measuring the growth curve of Agrobacterium C58,it was determined that the bacterial solution cultured for 12-18 hours was selected to prepare the infectious solution.The genetic transformation system of cucumber was optimized from the aspects of co-cultivation temperature,co-cultivation time and the concentration of Agrobacterium infection solution.The results showed that when the co-cultivation temperature was 23 ℃,the explant regeneration rate reached the highest 31.70%,and the positive induction rate reached the highest 2.26%;the concentration of the infecting solution and the co-cultivation time were combined to be treated.When it was 0.1 and the co-cultivation time was 7 days,the explant regeneration rate reached the highest 40.13%,while the positive induction rate of explants under different treatments was not significantly different.In summary,in this study,an optimized genetic transformation system was obtained in which the inoculum was selected from the bacterial solution cultured for 12-18 hours,and the co-cultivation temperature was 23 ℃.2.Creation and rapid identification of cucumber T-DNA insertion mutantsUsing the optimized cucumber genetic transformation system,pGreen0029 plant expression vector was used as a T-DNA insertion vector to create a cucumber T-DNA insertion mutant.To plants were identified by GFP fluorescence identification,PCR and TAIL-PCR.GFP fluorescence observation was used to screen for positive plants at the stage of explant differentiation,three positive shoots expressing GFP signals were screened out of 2978 transformed explants.PCR identification results showed that the T0 transformed plants had integrated vector sequences.T-DNA flanking sequence amplification using the TAIL-PCR method,the T-DNA insertion site of the mutant T-DNA-PG-1 was initially determined.Designing specific primers based on the T-DNA insertion site further confirmed the accuracy of the T-DNA insertion site.The comprehensive results showed that the pGreen vector was transferred into the cucumber genome,and the insertion position of T-DNA in the cucumber genome was determined by the T-DNA flanking sequence amplification.