Optimization Of Genetic Transformation System And Construction Of T-DNA Insertion Mutants In Cucumber

Cucumber is the first horticultural crops of which genome sequencing has completed.As a result,functional genomics becomes the key point of cucumber research.One of the most direct and effective ways of studying gene function is to construct mutant library,and then isolate gene and analysis of gene function through mutant.Insertional mutagenesis is the main method to create plant mutant library.With Agrobacterium-mdidiated transformation system of cucumber gradually developing and T-DNA insertion site identification methods enciched and improved,T-DNA insertional mutagenesis has been widely used in construction of plant insertion mutant library.Construction of cucumber T-DNA insertion mutants has great significance in the study of cucumber gene function,and aslo provies strong reference to the functional genomics research of:other cucurbitaceae plants.T-DNA insertional mutagenesis is based on Agrohacterium-mediated genetic transformation system.Therefore,efficient and stable cucumber genetic transformation system is the pre-condition.There are factors affecting the genetic transformation efficiency including cucumber genotype,explant,medium components,strain type,vector type,bacterial concentration,infection time,pre-culture time,co-culture time and selection pressure and so on.Medium components mainly involved inorganic nutrients,organic nutrients,growth regulating substances,curing agents and other additives,etc.Currently,cucumber genetic transformation efficiency is still low.Researchers had carried out a lot of research on optimization of genetic transformation system from growth regulating substances,culture conditions and selection pressure,but less research in curing agents,antioxidants and organic additives.This research optimized Agrobacterium-mediated genetic transformation system with changing medium curing agent and adding antioxidant and organic additive into medium.These improved the transformation efficiency.On this basis,we gained cucumber T-DNA insertion mutant with plant expression vector pROK2 used to generate insertion mutant as T-DNA insert vector transferred cucumber.This research laid an important foundation for the construction of cucumber insertion mutant library and studying cucumber gene function.Concrete contents and results are as follows:1.Optimization of Agrobacterium-mediated genetic transformation system of cucumberBased on previous Agrobacterium-mediated genetic transformation system built by our lab,the most suitable selection pressure concentration of kanamycin was confirmed by setting selection pressure gradient with cotyledon node of cucumber cultivars 'Changchun mici'.At the same time,the Agrobacterium-mediated genetic transformation system of cucumber was optimized with changing medium curing agent and adding antioxidant and organic additive into medium in different culture stage.The results indicated that the selection pressure concentration of kanamycin of 'Changchun mici'was 100 mg/L.The resistant buds induction rate on medium gelled with gellan gum was increased by 28.21%compared with those on medium gelled with agar.Adding 50 mg/L a-LA into co-culture medium could make the resistant buds induction rates of 'Changchun mici' up to 66.67%and number of shoots per explants up to 1.32.Similarly,when adding 1.5 g/L CH into selection culture medium,the resistant buds induction rate and number of shoots per explants of 'Changchun mici' were the highest,67.15%and 1.42 respectively.In conclusion,this article gained the optimized genetic transformation system that was using gellan gam as medium curing agent,then adding 50 mg/L a-LA into co-culture medium or 1.5 g/L CH into selection culture medium.The resistant buds induction rates gained from the optimized genetic transformation system were higher than that of the previous study of our lab which was 44.40%.2.Construction of Cucumber T-DNA Insertion MutantsWith the cotyledon nodes of cucumber cultivars 'Changchun mici' as the explants and the optimized Agrobacterium-mediated genetic transformation method,transfer T-DNA insert vector pROK2 into cucumber.PCR and dot blotting were used to identify the T0 and T1 ransgenic plants.Investigate and statistics the phenotypes of T1 plants used 'Changchun mici' as control.PCR detection results of T0 plants indicated that 35S promoter and NPT-II gene sequence were integrated into 14S1 line and 14S2 line.55 T1 plants were gained from 14S1 self-pollinated and 12 of them could amplify 35S promoter and NPT-? gene sequence.The results of dot blotting detection of the 12 plants and 11 plants randomly selected from the other T1 plants by dig labeled 35S probe and NPT-? probe were that 14S1-27 and 14S1-34 had 35S and NPT-? hybridization signals.Traits investigation showed the phenotype of T1 generation from 14S1 self-pollinated had no difference with'Changchun mici'.In a word,pROK2 recombinant plasmid was integrated into 14S1 successfully.Results of PCR and dot blotting detection of 14S1 selfed progeny were further proved that 14S1 was transgenic plants that is T-DNA insertion mutant.