Establishment Of YL-1 Genetic Transformation System And Functional Identification Of Flower Color Gene BolC.cpc-1 In Cabbage

Cabbage?Brassica oleracea L.var.capitata?is one of seven subspecies of Brassica oleraceacultivatedwordwidelyasanimportantvegetablecrop.Agrobacterium-mediated genetic transformation of cabbage started in the 1980s.However,the transformation efficiency is still very low and the stability is still very poor.Petal color is an important trait of plants which can be used as a visual signal to attract insects to pollinate and protect plants from damage.Petal color gene in Brassica oleracea was previously fine mapped and a candidate gene named BolC.cpc-1 was predicted.In order to identify the function of BolC.cpc-1,the overexpression vector of BolC.cpc-1 was constructed.Meanwhile,the genetic transformation system was optimized using yellow flower cabbage inbred line YL-1as a material.On the basis of the established genetic transformation system,YL-1 was transformed by Agrobacterium tumefaciens.The PCR detection,phenotypic observation and RT-PCR expression analysis were carried out.The main results are as follows:?1?The overexpression vector pBWA?V?BS-BolC.cpc-1 was successfully constructed.Primers were designed based on the previously cloned candidate gene sequences,and the reverse transcribed cDNA was used as a template to amplified and obtain a full-length 1791 bp CDS sequence.Next,the gel-recovered product and the vector were digested by Eco31I and the target fragment was ligated into the empty plasmid by using a one-step homologous cloning kit to obtain the recombinant vector pBWA?V?BS-BolC.cpc-1.?2?The high efficiency transformation receptor YL-1 was screened and an efficient transformation system of it was established.The effects of genotype on regeneration frequency were studied using hypocotyls and cotyledons of four Brassica oleracea L.materials.The results indicated that inbred line YL-1 was the best transformation receptor.Then,the effects of explant type,light intensity,seedling age,herbicide concentration,Agrobacterium tumefaciens concentration,acetoconone concentration and co-culture temperature on YL-1 adventitious bud regeneration were studied using hypocotyledonary axis and cotyledon with petiole.The results showed that there was no significant regeneration ability difference?P<0.5?between the cotyledon and hypocotyls,all that could be used for genetic transformation.The frequency of adventitious bud induction was the highest with 4000 lx light intensity using 5 days age seedling.8 mg.L^-1 herbicide Baster concentration could completely stopped adventitious shoot regeneration,so the concentration was selected as the lowest screening concentration.When the explants were inoculated by Agrobacterium with OD₆₀₀=0.3 for 8 min,the regeneration frequency of herbicide-resistant buds was the highest.The effect of acetosyringone on adventitious bud regeneration of YL-1 is little,and the concentration was too high and might even decrease the adventitious buds.Finally,the explants placed at a co-cultivation temperature of 25°C preferred to obtain a high regeneration rate after the Agrobacterium infection,.?3?The transgenic plants were obtained and PCR amplification was carried out.Based on the established genetic transformation system,genetic transformation of cabbage YL-1 was carried out by Agrobacterium-mediated method.At present,47resistant plants were obtained.The transgenic plants were screened using the target gene-specific primer Pboc-1 and the herbicide marker Bar-2.The results showed that the 12 resistant seedlings sequences with the same band size as the control could be detected in,demonstrating that the gene has been integrated into the YL-1 genome.?4?The flower color of transgenic plant turned yellow into white,which identified the function of BolC.cpc-1 gene in flower color variation.The petal showed different yellow degrees,and one of which showed complete white color.RT-PCR analysis of the expression of BolC.cpc-1 in the petals of transgenic plants,recipient plants and white petal parent plants revealed that BolC.cpc-1 did not express in recipient plants while the highest expression level was detected in white petal parent plants.The expression level of YF-2 was almost the same as that of white petal parent plants.In other transgenic plants,the color was more white when the gene expression level was more high.This result proved that the BolC.cpc-1 gene indeed controls the changes of petal color incabbage.