Cloning And Functional Analysis Of Red Zaosu Pear Anthocyanin Biosynthesis Related Genes

Pear is one of the main fruit in the world,whose pulp is crisp and juicy,sweet and sour. Itis very popular among Chinese people. Skin colour is an important quality parameter. The redpear is very beautiful with antioxidant anthocyanin, so it owns very high economic value andmarket competitiveness. Red pear breeding has become one of the main goals of pearbreeding at home and abroad. On the former basis of constrction of SSH cDNA library,Wecloned anthocyanin biosynthesis related genes and analyzed their function,in order to lay afoundation for molecular breeding of red pear.The main results were as follows:1.To analyze different gene expression patterns in anthocyanin biosynthesis pathwayof‘Zaosu’pear and its red mutant,five cDNA coding regions of key genes were isolated frompeels of zaosu and the red mutant using homologous cloning.In ‘Zaosu’ pear and its red mutant, Chalcone synthase（CHS）has an ORF of1170bpencoding a polypeptide of389amino acids.There are eleven different bases and zero differentamino acids between them.The homology analysis demonstrated that CHS shared highsimilarities of nucleotides and deduced amino acids between them over99.06%and100%;Dihydroflavonol4-reductase（DFR）has an ORF of1044bp encoding a polypeptide of347amino acids.There are nine different bases between them. The homology analysisdemonstrated that DFR shared high similarities of nucleotides and deduced amino acidsbetween them over99.14%and99.42%; UDP glucose:flavonoid3-O-glucosyltransferase(UFGT）has an ORF of1440bp encoding a polypeptide of479amino acids.The homologyanalysis of UFGT demonstrated that similarities of nucleotides and deduced amino acidsbetween them were100%; Basic helix loop helix（bHLH）has an ORF of1947bp encoding apolypeptide of648amino acids.The homology analysis of bHLH demonstrated thatsimilarities of nucleotides and deduced amino acids between them were100%;MYB10has anORF of735bp encoding a polypeptide of244amino acids.The homology analysis of MYB10demonstrated that similarities of nucleotides and deduced amino acids between them were100%. Sequence alignment and phylogenetic analysis revealed that these genes shared highsimilarities with genes from other plants.2.We used the Real-time PCR method to determine the relative expression of fourstructural genes CHS、F3H、DFR、UFGT,and two transcription factors bHLH、MYB10indifferent plant tissue of ‘Zaosu’pear and its red mutant.Relative real-time PCR analysisindicated that most genes of the red mutant showed higher transcription levels than zaosu inleaves,leafstalks,flowers,fruitlets,flowerstalks.Among these expression differences,UFGT andMYB10were distinct.3.By inserting ORF sequence of target gene into the plasmid pBI121under the control ofCaMV35S promoter, the F3H、DFR、UFGT、MYB10and bHLH plant expression vectorswere constructed successfully.4.PbMYB10was transferred into tobacco via agrobacterium mediation. Ninety-sixregenerated plants were obtained.Eight transgenic plants were selected randomly to beconfirmed by PCR and five positive plants were confirmed.