| Cucumber(Cucumis sativus L.)is an important facility-cultivated vegetable in our country.Drought has caused its growth and development to be delayed,its yield has seriously decreased,and its fruit quality has deteriorated.Genetic transformation verification is an important method for studying gene function and modern molecular breeding today.Establishing a stable cucumber genetic transformation system can provide technical support for cucumber functional genomics research using overexpression and other techniques.In this study,the cucumber PIP2;4 gene was taken as the research object,the PIP2;4 gene was cloned and bioinformatics analysis and genetic transformation studies were carried out.The main results are as follows:(1)Bioinformatics shows that CsPIP2;4 is 852 bp long and encodes 283 amino acids.CsPIP2;4 protein is hydrophobic,has no signal peptide,and has a transmembrane structure.It is an intrinsic protein located in the plasma membrane.The coiled and ?-helix structures are dominant,with typical aquaporin structural domains,and belong to the MIP superfamily;the protein homology ratio shows that CsPIP2;4 is closest to melon PIP2;7.The q PCR results indicated that the expression of CsPIP2;4 was induced by drought and may be involved in the regulation of drought stress.(2)Constructed the expression vector p CAMBIA 2300s-PIP2;4 regulated by Ca MV 35 S promoter;the selective pressure screening of adventitious bud and root induction of cucumber '9930' was carried out,and the optimal screening concentration was 150 mg/L Kan,while The optimal concentration of Xintai Mipin' rooting medium is 100 mg/L Kan;sterilization of the explants after co-cultivation can inhibit the proliferation of Agrobacterium and increase the survival rate of the explants;it can be significantly improved without pre-cultivation Induction rate of resistant buds of cucumber;When the p H of differentiation medium is 5.6,it can increase the regeneration bud rate of cucumber explants;The optimal rooting concentration of cucumber'Xintai Mithorn' tissue culture seedlings is 0.5 mg/L IBA;Rooting medium Compared with the control group,the medium-added MES showed a very significant difference in POD activity in the root system and a significant difference in the leaves.Using the optimized genetic transformation system,using "Xintai Mipin" as the material,overexpressing PIP2;the conversion rate of 4 cucumber plants was 4.13%;using "9930" as the material,overexpressing PIP2;4 conversion rate of cucumber plants 2.89%.This experiment optimized the cucumber genetic transformation system and obtained the overexpressed PIP2;4 cucumber transgenic lines,which provided the basis for the optimization of cucumber genetic transformation system and the verification of gene function. |
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