The Molecular Regulation Mechanism Of GGP Gene Related To Vc Concentration In Fruits

L-ascorbic acid(AsA)is the most important compound influencing the fruit quality,the biosynthesis contributes the main AsA in plants.GDP-L-galactose phosphorylase(GGP),as the main limited enzyme of AsA biosynthesis in plants,plays a crucial role for AsA concentration.In this thesis,Actinidia eriantha and Actinidia rufawhich showed significant difference on AsA concentration were used as the material,we analyzed their sequences and activities of GGP promoters;Comparison with the AMR1 gene from Arabidopsis in apple genome database,MdAMR1.1 and MdAMR1.2 were cloned from applecDNA,and analyzed their relationship with AsA concentration;By screening the cDNA library of yeast one-hybrid,potential transcription factors were selected.The results as following:1.The relative expression of GGP1 gene showed higher correlation with AsA concentration in Actinidia eriantha,Comparison of the hybrid progenies of Actinidia eriantha and Actinidia rufa,AsA concentration showed higher relationship with Actinidia eriantha.The separation of GGP promoters in the hybrid progenies showed that GGP1 and GGP2 were linked,and the promoters of GGP1 genes in Actinidia eriantha may have relationship with AsA concentration.The results of a further investigation about the difference of GGP1 allelic promoters in Actinidia eriantha and Actinidia rufa showed,comparison with Actinidia rufa,the promoters of AeGGP1(Actinidia eriantha)lost 217 bp and 215 bp in length respectively,but had higher activities than the other two promoters.There occurs some difference in the activities of GGP1 allelic promoters in Actinidia rufa.These results indicated that the inheritance of GGP1 promoters had significant roles in determining AsA concentration.Also,the investigation on the relationship with uORF region and AsA concentration showed the genome editing in this region may improve the AsA concentration.2.Comparison withAMR1 gene which negatively regulated AsA concentration of Arabidopsisin apple genome database,MdAMR1.1 and MdAMR1.2 were cloned from cDNA of ‘GaLa’ apple.The analysis showed that MdAMR1.1hadsignificantly negative correlation with AsA concentration during the whole developmental stages of apple fruit.By transforming these two genes in wild type and mutant Arabidopsis,overexpression of these two genesdecreased AsA concentration obviously,and MdAMR1.1 was better for the AsA decrease in Arabidopsis.In order to illustrate the regulation approach of MdAMR1.1,the promoter of MdAMR1.1was cloned from apple,and constructed the recombinant vector with this core sequence for yeast one-hybrid.By screening the cDNA library of yeast one-hybrid,eight potential genes were screened,they were AMR1-1,AMR1-5,AMR1-6,AMR1-7,AMR1-14,AMR1-16,AMR1-22 and AMR1-27,these results laid a solid foundation for the further study of the transcriptional regulation.