Preliminary Study On Transformation Of Cucumber With RNAi Fragment Of CsMADS21 Gene Mediated By Agrobacterium

Cucumber（Cucumis sativus L.）is an important vegetable crop.The AP1-FUL MADS-box gene family plays an important regulatory role in the process of plant growth and development.Cs MADS21 belongs to it,and its function in cucumber needs to be further confirmed.In this study,four genotypes of cucumber were used as materials to discuss the genetic transformation system from genotype,aspects of explants,hormone concentration,hygromycin and timentin concentration.At the same time,the p CAMBIA1305-Cs MADS21RNAi vector was constructed and transformed into cucumber cotyledon nodes by Agrobacterium-mediated method.The resistant plants were detected by hygromycin screening,molecular detection and tissue staining.Phenotypic analysis of T₀ generation transgenic plants was carried out.The main results were as follows:1.Construction of p CAMBIA1305-Cs MADS21RNAi vector.After comparative analysis,a 200 bp conserved sequence of Cs MADS21 gene was selected and inserted into the first intron（199 bp）of the potato GA20 oxidase gene forward and reverse to construct a 599 bp interference fragment.The p CAMBIA1305-Cs MADS21RNAi vector was constructed by Bam H I-Xba I double enzyme digestion and ligated into p CAMBIA1305-GFP vector.After verification by Bam H I-Sac I digestion,a fragment about 1400 bp（interference fragment+d35S promoter）was obtained,indicating that the p CAMBIA1305-Cs MADS21RNAi was successfully constructed.2.Study on the genetic transformation system of cucumber.The genetic transformation system of cucumber was researched from genotype,aspects of explants,seedling state,hormone concentration,hygromycin and timentin concentration.The results showed that the regenerative ability of inbred line 2014-3 was significantly higher than others.The best explants were the cotyledon nodes with erect cotyledon but not yet expanded at seedling age of about 3 days;The optimal hormone concentration was also different for different genotypes.The optimal bud induction medium for inbred line 2014-3 was 2.0 mg/L 6-BA+0.5 mg/L ABA+1.5 mg/L Ag NO₃,and the bud regeneration rate reached 75%;The optimal bud induction medium for inbred line 2014-5 was 1.5 mg/L 6-BA+0.5 mg/L ABA+1.5mg/L Ag NO₃,and the rate reached 66.67%;10 mg/L Hyg was used in bud induction and rooting stages.0.1 mg/L 6-BA was used in elongation and 400 mg/L timentin was used to inhibit the growth of Agrobacterium after infection.3.Genetic transformation of cucumber with p CAMBIA1305-Cs MADS21RNAi vector.The p CAMBIA1305-Cs MADS21RNAi was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method.The cotyledon nodes were inoculated in Agrobacterium solution with OD₆₀₀=0.7-0.8 for 10 min.After co-cultivation for 2 days,and then cultured in the optimal bud induction medium.The non-transformed cells were screened by 10 mg/L Hyg,a total of 46 resistant plants were obtained,including 11 resistant plants in inbred line2014-2,9 resistant plants in inbred line 2014-3,4 resistant plants in inbred line 2014-4 and22 resistant plants in inbred line 2014-5.4.Detection of transgenic plants.The resistant plants were amplified by PCR using Hyg and GUS primers.GUS histochemical staining and GFP fluorescence detection was performed on different organs of transgenic plants.The results showed that the corresponding bands were obtained after PCR amplification.After GUS staining,the pistil stigmas of transgenic plants were slightly blue,the anthers of male flowers were dark blue,other organs did not appear blue.Fluorescence detection showed that there was clear green fluorescence in the pollen sacs of transgenic plants.It indicated that the target gene had been successfully transformed into cucumber.The conversion rate of inbred line 2014-2 was0.35%,inbred line 2014-3 was 0.98%,inbred line 2014-4 was 0.25%,and that of inbred line2014-5 was 1.25%.5.The observation of early phenotype of transgenic plants.The phenotypes of T₀ generation plants were statistically analyzed.The results showed that the main stem naturally withered,the main stem height and average internode length decreased,the number of lateral branches increased,and the fruit length reduced.