Cloning Of Ppo Gene Promoter From Litchi (Litchi Chinensis Sonn.) And Primary Functional Characterizaion

The development of litchi industry is limited by postharvest pericarp browning, Research on postharvest physiology proved that enzymatic browning was the major reason for the pericarp browning and polyphenol oxidase played a main role in the process. Our previous research showed that expression of PPO gene was related to the peel browning process. The promoter of PPO gene was cloned; The core elements and cis-action elements of the promoter were analysised; Discusses the different of PPO gene promoter sequence in different litchi The function of promoter of PPO gene in Litchi's pericarp were identified by combination with constructions of plant expression vectors, which will provide another way of resolving browning of pericarp in Litchi by modification of cis-action elements in promoters to reduce effectively expression of PPO gene.The promoter sequence1048bp of PPO gene in feizixiao Litchi's pericarp was cloned. The promoter of PPO gene in Litchi's was78%homologous compared with peach in the nucleotides by NCBI-BLASTN. The sequence was analysised with Plant CARE software, the results showed that the predicted starting site of transcription was the base A at the-102bp; there were TATA-Box and CAAT-Box at-149bp and-203bp; there was a possible basic promoter area between-244and-194bp. Some conserved elements of core-promoter such as TATA-Box, CAAT-Box, G-Box, ect. and some cis-action elements such as GATA-motif,P-box,TCA-element ATGCCAAT-moti,ARE etc were found in promoter, that the cis-action elements perhaps were related to tissue specificity of promoter of PPO gene in Litchi's pencarp.The promoters of PPO gene sequence were992bp and979bp in ziniangxi and wuhe Litchi's pericarp by homology cloning. Sequence alignment analysised that the PPO gene promoter sequence homology was98%for three varieties, the single nucleotide polymorphisms (SNP) were22, including transversion was19, conversion was2and insertion (deletion) was1; and A T bases inserted. The different type and quantity of cis-action elements were existenced because of the single nucleotide polymorphisms. The enzymes Sal I and Xba I digests the promoter fragment, getting different lengths of enzyme product, to confirm the SNP in the promoter sequence.The CaMV35S promoter in the plant expression vector PBI121was replaced by the deletion fragments of promoter sequences. The different lamina,pericarp and seed of Litchi were transformed with the constructed plasmids (PPO-PBI121-1 PPO-PBI121-2and PPO-PBI121-3) by bombardment transformation, respectively; transient expression of GUS gene was observed by histo-chemical dyeing. The results showed that observed the blue spots by transformed with these plasmids, explaining the promoter PPO gene can turn on the GUS gene expression in lamina and pericarp; but the leaves and seed tissue were not observed blue spots, explaining the promoter PPO gene can't turn on GUS gene expression in seed.