Cloning And Functional Identification Of The Promoters For Genes Encoding Polyphenol Oxidases In Nai (Prunus Salicina Lindi.var.cordata J.Y.zhang Et Al)

The promoter of PPO gene in Nai's pulp was cloned by genomic walking,and core elements and cis-action elements of the promoter were analysized with PlantCare software;The functions of promoters of genes encoding PPOs in Nai's leaves and pulps were identified by combination with constructions of plant expression vectors containing different deleting fragments of the promoters,bombardment transformation and histo-chemical dyeing of product of transient expression for GUS gene,which will provide another novel way of genetic improvement for further resolving browning of pulp in Nai by modification of cis-action elements in promoters of genes encoding PPO and to reduce effectively expression of PPO genes.The main results were as follows.1.The promoter of PPO gene in Nai's pulp was cloned by genomic walking.The promoter of PPO gene in Nai's pulp was cloned by two methods of genomic walking i.e.adaptor-mediated PCR and thermal asymmetric inter-laced PCR(TAIL PCR).The results showed that TAIL PCR was superior to adaptor-mediated PCR; that the amplifying product of TAIL PCR was about 700bp,the sequencing results showed that length of product of TAIL PCR was 768bp which covered the whole sequence of amplifying product of adaptor-mediated PCR whose length was 495bp. The sequence of TAIL PCR product analysized with PlantCare software,the results showed that there was a possible basic promoter area between 585bp and 635bp in the promoter of PPO gene in Nai's pulp,that the predicted starting site of transcription was the base A at the 625bp;and that some conserved elements of core-promoter such as TATA-Box,CAAT-Box,G-Box,etc.and Some cis-action elements such as I-box, G-Box,GATA-motif,AT-rich sequence,MBS etc.were found in the sequence of promoter,that the cis-action elements perhaps were related to tissue specificity of promoter of PPO gene in Nai's pulp which were 78%homologous compared with that of PPO gene in peach in the nucleotides,therefore,it can be concluded that the cloned regulatory sequence in 5' terminal of PPO gene had a characteristic of a common plant promoter. 2.The functions of promoters of genes encoding PPOs in Nai's leaves and pulps were identifiedThe functions of promoters of genes encoding PPOs in Nai's leaves and pulps were identified by combination with constructions of plant expression vectors containing different deleting fragments of the promoters,bombardment transformation and histo-chemical dyeing of product of transient expression for GUS gene.The 35 S promoter in the plant expression vector- pCAMBIA1301 containing GUS gene was replaced forward by the deletion fragments of regulatory sequences in the 5/ terminal of PPO genes with different lengths in Nai's leave(1160bp,1037bp,935bp and 825bp)and pulp(569bp,314bp and 215bp),respectively;the leaves and pulp of Nai were transformed with the above constructed plasmids by bombardment transformation,respectively;transient expression of GUS gene was observed by histo-chemical dyeing in Nai's leave and pulp.The results showed that expression of GUS gene in the plasmid containing 569bp deleting fragment of the promoter of PPO gene in Nai's pulp was stronger than that of GUS genes in two plasmids containing 314bp and 215bp,the whole pulp transformed was dyed into blue,but less than 5% of pulp transformed by plasmids containing 314bp was dyed into blue for lower expression of GUS gene,and the pulp transformed by plasmids containing 215bp was not dyed into blue for no expression of GUS gene;the leave was transformed with the above three plasmids containing three different length of deleting fragments of promoter in pulp by bombardment transformation,the tissues transformed were not dyed into blue for no expression of GUS gene in the three plasmids,it was concluded that was the promoter of PPO gene in Nai's pulp was a fruit-specific promoter which only works in pulp and did not work in other tissues in Nai and the length of basic promoter ranged between 324 and 569bp;that the leaves and pulps were transformed with four plasmids containing different lengths of deleting fragments of promoter in leave(1160bp,1037bp,935bp and 825bp)by bombardment transformation,and the whole transformed tissues of leaves and pulps were all dyed into blue for expression of GUS gene,it was primarily concluded that the promoter of PPO gene in Nai's leaves was non-tissue-specific promoter because it both works in leaves and pulp,and that the length of basic promoter must be shorter than 825bp,and the accurate length of the basic promoter was further determined by constructions of plant expression vectors containing different deleting fragments of the promoters and bombardment transformation.