Cloning Of Polygalacturonase (PG) Gene In Peach And Construction Of The Antisense Expressing Vector

Peach fruit,a climacteric fruit,is very difficult to be storaged and transported because it is very to be soften after harvested.The tradditional storage,which not only need high charge but also have a limited fresh period,can not solute the softing problem of peach fruit ultimately.It has hope to solve the problem by tranditional breeding,but peach is perennial plant so breeding time is very long and germ resource has limitation.So it is hard to get breakthrough in short time.The softening and mature of peach fruit related to degradation of a large number of pectin,which mainly due to the role of Polygalacturonase(PG).To silence the expression of PG which lead peach fruit soft by antisense gene technology may increase the storaging function of peach fruit and obtain new varieties of peach.This technology has been succeed in tomato,It laid down a solid foundation and provided beneficial information for solving the difficult softing problem of peach fruit using antisense gene technology.The objective of the study was to obtain the complete sequence of PG gene and construct the plant expression vector.The study laid down a solid experiment foundation for further explant transformation of peach varieties with the PG gene to improve the quality of peach fruit.The results were as fallows:(1) Total RNA was extracted from mature fruits of Baifen peach by Trizol,improved Trizol method.The improved Trizol method is the better method in the RNA extract.By using the specific primers designed from the PG gene in GeneBank,a fragment about 1188bp with an open reading frame of 393 amino acids was produced by reverse transcription and polymerase chain reaction.The amplified fragment was cloned into pGEM-T vector and it was identified by sequence analysis after PCR and restriction enzyme.The cDNA nucleotide sequence and deduced amino acid sequence were highly homologous to that of the reported peach PG cDNA,the results showed that this fragment was PG gene fragments of Peach.(2) Plasmid pGEM-PG and pBI121 were double digested with restriction endonucleases BamH I and Sac I to retrieve the PG gene and the linear vector without gus gene.Thus the linear pBI121 and the PG had reversely matching ends.After ligating the two fragments DNA,transformed into E.coli cells.Indentification of restriction map by BamH I / Sac I digestion indicated that 1.2kb fragment was obtained.Thus, the intermedial plant expression vector pBI-aPG containing reverse PG gene was successfully constructed.It laid down a solid foundation and provided beneficial information for further transformation of peach varieties. (3) The recombination plasmid pBI-aPG containing PG gene was transferred into Agrobacterium tumefaciens LBA4404 by direct DNA transfer method.Identified with PCR detection,the transformation of the plasmid was successful.