| In this study,‘Pinot Noir',‘Red Globe',‘Youngle',‘Heshi Seedless',‘Ruby Seedless',‘Flame Seedless' and ‘Thompson Seedless' seven European grapes with different seed sizes,were used as material to research the expression pattern of ABC transporter ABCG20 gene in the ovule development;qRT-PCR was used to analyze the gene's response to plant growth regulator treatment of ovules and leaves;The expression level of the VvABCG20 gene in endosperm,seed coat and ovule of ‘Pinot Noir' grapes was used to analyze the tissue specificity of the VvABCG20 gene;The subcellular localization analysis of grape ABCG20 protein and the transport functional analysis in grape suspension cells and callus were performed.The main results are as follows:1.The expression of VvABCG20 gene in ovule development of seven grape varieties with different seed sizes was analyzed by qRT-PCR,it was found that the expression level of VvABCG20 gene was higher in grape ovules with larger seed size than grape varieties with smaller residue.The seed coat and endosperm of 30-and 40-day unhardened seeds of ‘Pinot Noir' grape varieties were separated,qRT-PCR analysis revealed that the expression of this gene in the seed coat was higher than in ovules and endosperm.It was found that the high expression gene VvABCG20 in grape ovule has the same response pattern to plant growth regulators GA3 and CPPU in the ‘Thompson Seedless' ovule,that is,with the extension of processing time,the gene expression was increasing;‘Pinot Noir' grape leaves were sprayed with the plant growth regulators ABA,6-BA,GA3,Eth and MeJA,fluorescent quantitative PCR analysis showed that VvABCG20 gene expression increased mainly in 12 h after treatment with the five plant growth regulators..2.The VvABCG20 gene and GFP fusion expression vector were constructed and transiently transformed into tobacco,and subcellular localization of grape ABCG20 protein was studied,confocal microscopy revealed that it was located in the cytoplasmic membrane.3.Using stem segments of ‘Thompson Seedless' and ‘Pinot Noir' grape as material,MS as the basical medium and plant growth regulators combinations containing 0.3 mg/L 6-BA and 2.0 mg/L NAA induced grape loose callus;A fast and stable ‘Thompson Seedless' cell suspension culture system was established under the optimized culture condition of B5 as basic medium,0.5 mg/L 6-BA and 1.0 mg/L 2,4-D plant growth regulators ratio and concentration,and 0.2% PVP.4.After treatment of cells with appropriate concentrations of cinnamic acid derivatives(phenolic acids),succinic acid and glycerol inducers,qRT-PCR analysis showed that the expression level of VvABCG20 gene was obviously increase after treatment with cis-ferulic acid,caffeic acid,coumaric acid,succinic acid and glycerol,even up to 30 times,indicating that the gene can responds to the induction of these five substances.5.High-performance liquid chromatography(HPLC)was used to determine the phenolic acids in the wild-type and VvABCG20 gene-overexpressed transgenic callus,and the results showed that the high expression of VvABCG20 gene could cause the changes of phenolic acid content in grape.The phenolic acid content of wild-type and transgenic callus treated with cis-ferulic acid,caffeic acid and coumaric acid was determined by HPLC,indicating that the treatment of cinnamic acid derivatives can increase the content of other phenolic acids in grape callus,and the content of other phenolic acids increased in the transgenic callus was higher than that of the wild-type.Therefore,it is preliminarily considered that the VvABCG20 gene may be involved in the biosynthesis and metabolism of grape phenolic acid. |
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