A Modified Protocol Of Agrobacterium-mediated Transformation And Map-based Cloning And Functional Analysis Of W Gene Controlling White Immature Fruit Color In Cucumber

Genetic transformation is an important technique for gene function study and crop im-provement.In cucumber,since the first report of transgenic plant in 1986,numerous efforts have been made to improve transformation protocol.Due to lack of experimental details,lim-ited access of seeds of cucumber genotypes and plasmid vectors,a reliable and reproducible protocol with acceptable transformation efficiency is not available for the cucumber research community.The immature fruit color is crucial commercial trait for cucumber consumption,while limited white skin commercial cultivars are just distributed in part of China.The chro-mosomal location and the molecular regulatory mechanism are still unknown,which restrict the marker-assisted selection breeding and transgenic breeding in cucumber.In this study,we revisited and optimized many technical details to develop a high efficient and stable protocol for cucumber transformation.Fine mapping and cloning of the target gene w controlling the white immature fruit.Identification and functional characterization of the candidate gene Ar-abidopsis two-component response regulator-like（APRR2）of w.The main results are as fol-low:1.A modified protocol of Agrobacterium-mediated transformation in cucumberThe Agrobacterium-mediated transformation using cotyledon as explants has been the major approach.The over-expressing plasmid vector pCAMBIA2301 harboring LITTLE LEAF gene（LL）was transferred to American slicing cucumber Poinsett76 mediated by Agro-bacterium strain AGL1.We found that the serrated and crescent wounds at the proximal part of the half cotyledons are good explants to induce shoots.The most of shoots were obtained when the explants were inoculated in the Agrobacterium suspension at OD₆₀₀ of 0.7-0.8 for 12min facilitated with vacuum infiltration.Adding a piece of filter paper on the co-culture me-dium could significantly decrease the Agrobacterium contamination during shoot initiation.After co-culture for 2 days,only the yellowish explants were transferred to selection medium containing 0.5 mg/L 6-BA,0.5 mg/L ABA and 100 mg/L kanamycin.No-chimeric shoots were excised and sub-cultured in selection medium for one more time.Only the seedlings with green leaves grown from antibiotic selection were transferred to 1/2 MS rooting medium supplemented with 50 mg/L kanamycin.Leaf sample form 5-true-leaf stage was taken for DNA extraction and PCR assays.Only PCR positive plants were kept growing for phenotyp-ing（T0）and production of progeny from self-pollination.Using this protocol and the LL as the test case,we got a transformation efficiency of 0.5%.2.Fine mapping of white immature fruit color gene w in cucumberTwo crosses of Q30×Q24 and WD3×B-2-2 from two green skin cucumber（Q30,WD3）and two white skin cucumber（Q24,B-2-2）were used in present study.By analyzing the in-heritance of white immature skin color in BC₁ and F₂,the white immature skin color was in-ferred to be controlled by a single recessive gene w.Initially,2971 F₂ plants were used to nar-row down the w gene into a 33.0 kb region.Four candidate genes were predicted and two of them,peroxidase superfamily protein（PSP）and APRR2,showed different expression level and amion acid diversity.To definitively map the sole gene,the mapping population was in-creased to 9497 F₂ individuals.Consequently,the w gene was narrowed into an 8.2 kb physi-cal region.Just one candidate gene APRR2 was located in this region.3.Map-based cloning,identification and characterization of APRR2Sequencing the full-length of DNA and cDNA of APRR2 allowed for the characteriza-tion of an allele,aprr2,encoding a truncated 101-aminoacid protein at the end of the gene due to a frame shift mutation and a premature stop codon at the ninth extron.To be a transcription factor related to fruit mature,homologous analysis of APRR2 in Arabidopsis indicated that these 101 residues are located in a G-box domain necessary for the protein function.The ex-pression patterns of APRR2 in Q30 were entirely consistent with the visual changes in green color intensity during fruit development.The microscopic and submicroscopic observation of the fruit pericarp revealed fewer chloroplasts,a lower chloroplast chlorophyll storage capacity and disorganised granum structure in Q24（white）than in Q30（green）.The single-base inser-tion（G）in the white color gene aprr2,which leads to a premature stop codon in transfor-mation,is hypothesized to has disabled the function in chlorophyll accumulation and chloro-plast development.4.RNAi of APRR2 in Poinsett76By using the transformation protocol established in the present study,the RNAi vector RNAi-LIC-APRR2 was constructed and expressed in Poinsett76 to interrupt the expression of APRR2 gene.Consequently,10 PCR positive plantlets were obtained and two of which showed the light green skin color in transgenic lines relative to the dark green in WT.The RNAi result certificated that APRR2 can regulate the formation of green skin color in cucum-ber.The present study established a transformation protocol with a high efficient and stable transformation efficiency of 0.5%.The function of the candidate gene APRR2 controlling white immature skin color was verified with forward and reverse genetics approaches.The achievement in this study will facilitate the marker assistant selection and cucumber breeding.