Cloning And Nucleotide Sequence Analyzing Of The Polygalacturonase Gene Promoter From Peach

Prunus persica ( L.) Batsch. is one of the oldest fruit tree in our country, which grows all over the temperate zone. Its fruit is climacteric and shows a series of changes such as colour, texture and flavour during its development and maturation, especially in the post-harvest ripening stage. It is quite easy to become soft, which always drops quality of the fruit as merchandise.In the post-harvest ripening stage polygalacturonase, which can degrade pectin substances and destruct cell wall structure, plays a major role and makes the fruit rapidly soften. Release and expression of polygalacturonase are strictly controlled by the gene, in which the promoter of polygalacturonase is an important cis-element of the gene expression and regulation and plays a key part for expression of polygalacturonase and its amount. In order to delay the post-harvest ripening of fruit and inhibit expression of polygalacturonase and further create the new variety, it is very important to study cis-element of the expression and regulation of PG gene, i.e. the promoter of it.The main purpose of this study is to clone the promoter of polygalacturonase and then to conduct an analysis for sequence of the promoter, and on the former bases construct a missing vector of expression and lay the foundation for later study of function.The main contents of the study include: to project three special primers according to principles of projected primers contrasting the reported sequence of peach polygalacturonase gene; to extract DNA by improving CTAB; to amplify and clone the 5′flank sequence of peach PG gene and then analyze it with the method of Tail-PCR; to compare BLASTn by landing NCBI; to submit orderly GenBank database; to amplify and clone missing fragments and construct a missing vector of expression. The result of this study indicates that homology between sequence cloned into the 3 'end and the reported sequence the 5 'end partial sequence of PG gene is about 96% and no homology exists the remaining 5 'end of the 906bp fragment in GenBank, which is proved that the fragment is PG gene 5 'flank sequence. Submit database, and accession number is FJ940722. Using Neural Network Promoter Prediction, PLACE and other software to conduct the sequence analyses including the basic promoter, and transcription initiation site and cis-acting element and the initiation codon, the result shows that the based promoter sequence exists in the location of 789bp-839bp and transcription initiation site is located at the base C of 828 bp, in which possibility is 0.98. Containing the rich cis-acting elements, besides the basic CAAT-BOX and TATA-BOX, sequence also includes other important components such as GATA-BOX, ARR1AT-BOX and ACGTATERD1 etc.On the base of analyses of the sequence, the thesis further projects the primer and obtains three missing fragments containing the important components of the sequence by amplifying, and recovers the fragments by replacing the 35S promoter in plant expression vector pBI121, and constructs of expression vector of the three missing plant vectors of expression, all of which lays the groundwork for such research on the future regulation of PG genes.