Genetic Transformation And Functional Analysis Of The Gene Controlling White Immature Fruit Skin Color In Cucumber

Fruit skin color is an important fruit quality trait for variety improvement in cucumber(Cucumis sativus L.).In this study,a white-skinned line H4 and a green-skinned cucumber inbred line Q1 were used as the female and male parent,respectively,to generate one F1 population via hybridization,then this F1 population were self-pollinated to produce an F2 population.We researched the internal physiological mechanism of the white immature fruit skin color trait in cucumber by chlorophyll content determination and chloroplast observation.Based on Dong Xu’s research(2015)of the gene controlling white immature fruit skin color in cucumber,the candidate gene region were narrowed by the method of map-based cloning.Then,quantitative real-time PCR analysis and TA cloning were used to identify the gene controlling the white immature fruit skin color in cultivated cucumber.Finally,genetic transformation of the candidate gene was performed.Results are summarized as follows:1.Chlorophyll Content Determination and Chloroplast Transmission Electron MicroscopyHigh levels of chlorophyll were detected in the green inbred line Q1 compared to the white inbred line H4,and the significant difference was found in the pericarp of fruit.Next,transmission electron microscopy was used to observe the number and ultrastructure of chloroplasts in the pericarp of the two parental lines.The chloroplast of the white inbred line H4 exhibited extensive internal vacuolization and premature senescence.The grana thylakoid of H4 lacked starch grains,and the internal structure of the plastid was fuzzy.The total number of plastoglobuli was decreased in H4 by comparing with Q1.Moreover,the chloroplast number and size of H4 were fewer and smaller than Q1.2.Marker Development and Genetic MappingPrevious study located the target gene,which controlling the white immature fruit skin color in cucumber,in the distal region of cucumber chromosome 3.63 new pairs of CAPS markers were developed,including 5 polymorphic markers in Q1,H4 and F1.Linkage analysis of these 5 pairs of polymorphic markers with the target gene of cucumber using 1655 white plants in the 7304 F2 individuals mapped the candidate gene.Two flanking markers,Q147 and Q193 located the gene locus to a 100.3 kb genomic DNA region,including 13 candidate genes.3.Quantitative Real Time PCR AnalysisQuantitative real time PCR analysis was performed to test whether the 13 candidate genes expression was altered between Q1 and H4.The relative expression levels of Csa3G904080 in the 8-and 13-day-old fruit pericarp were 1.86-fold and 2.42-fold,respectively;the relative expression levels of Csa3G904110 in the 8-and 13-day-old fruit pericarp were 1.59-fold and 6.15-fold,respectively;the relative expression levels of Csa3G904130 in the 8-and 13-day-old fruit pericarp were 5.34-fold and 9.19-fold,respectively;the relative expression levels of Csa3G904140 in the 8-and 13-day-old fruit pericarp were 5.01-fold and 22.63-fold,respectively.For another 9 candidate genes,the relative expression level in fruit skin was too low.The results of the tissue expression revealed that Csa3G904080 was highly expressed in root and leaf;Csa3G904110 was highly expressed in root,stem and pericarp;Csa3G904130 was highly expressed in pericarp and root;While Csa3G904140 was highly expressed in the pericarp of fruit rather than other tissues.4.TA Cloning SequencingBy comparing candidate gene coding sequence between Q1 and H4,three SNP mutations in exon were detected within the Csa3G904080 gene,which led to three amino acid changes.There existed two same sense mutations within the Csa3G904110 gene,and the c DNA sequence of Csa3G904130 in Q1 was the same as that of H4.There was one single-nucleotide insertion in the ninth exon resulting in a premature stop codon within the Csa3G904140 gene.All SNP mutations in Csa3G904080 and Csa3G904140 also existed in g DNA,the possibility of RNA editing was eliminated.In addition,the mutation in the Csa3G904140 gene was consistant with the phenotype of 17 green/white germplasms,while the mutations in the Csa3G904080 gene were not consistant with the phenotype of 17 green/white germplasms.Therefore,Csa3G904140 was likely to be the candidate gene controlling the white immature fruit skin color in cucumber.5.Genetic Transformation of Csa3G904140The Csa3G904140 gene was transferred into H4 using agrobacterium-mediated method,a total number of 52 regeneration plants was obtained.Among them,17 plants were positive by PCR detection.Quantitative real time PCR analysis showed the gene expression level of some transgenetic plants was increased,but phenotypic observation found that the fruit skin color of the transgenetic positive plants only slightly turned green and the change was not significant.