Cloning Of Cherry PGIP Gene And Its Promoter

The Polygalacturonase inhibiting protein (PGIP) play a very important role in fruit ripening during fungal invasion, PGIP gene can be used in fruit tree gene engineering. PGIP gene expression can also be induced by wound and elictor, the promoter of PGIP gene is induciable and organ specific, and it can be induced by wound, pathogens and elictor. Cloning and sequencing the PGIP promoter is of great value for studying the PGIP gene expression pattern in ripening and for use of the promoter in fruit biotechnology.With the material of disease resistance cherry stock cultivar (Prunus mahalebL.), a pair of PGIP specific primer was designed to amplify the PGIP gene. By the reverse transcription polymerase chain reaction(RT-PCR) method, a fragment about 1000 bp was cloned and sequenced. The sequencing result showed that the fragment was 105^1 bp long, including a open reading frame(ORF). The open reading frame included 990 bp, encoding 330 anmino acids. Homology analysis showed that the sequence were 97. 2%, 83. 4% and 83. 6% homology to the cDNA sequence of apricot, pear and apple repectively. The possible encoding protein were 96. 7%, 85. 2% and 85.2% homoly to that of apricot, pear and apple. The result implified that we got the right PGIP cDNA fragment of Prunus mahaleb L. The sequence was loged in the Genbank by the accession NO. AF263465.In order to get the promoter of PGIP gene, the PGIP DNA sequence and the intron and extron must be known when design the primer. By the PCR method, we got a fragment about 1200 bp, sequencing and homoly analysis result showed that the fragment was 1192 bp's long, and was 94.1%, 82.4% and 75.2% homoly to the cDNA sequence of apricot, pear and apple respectively, it also showed the sequence might have a intron. Comparison of the PGIP DNA and cDNA sequence showed that the PGIP DNA seqence have two extrons interrupted by a 147 bp's intron, the first extron indued 581 base pair, the second is 464 bp. The intron located in the middle of the fragment and it is not too larger to amplify through.Inverse PCR , RAPD/anchored PCR and a modified nested PCR method were used to amplify the PGIP DNA upstream sequence. Comparison showed the inverse PCR method was complex in template prepair and was difficult to aquire the interstedfragment, the anchored PCR was difficult to screen the RAPD primer and set the anealing temperature, the modified nested PCR was more successful and a 2200 bp fragment was aquired by this method. Sequencing results showed the fragment was 2268 bp including partial PGIP cDNA region of 198 bp, sequence analysis showed there was a TATA box at 1963-1969bp, 148bp before the start codon; a CAAT box at 1886-1889, 225bp before the start codon; and a G box at 1911-1919 in the fragment, a 29 bp direct repeat unit was also found at 1251-1279,1295-1323. The sequence had the character of a plant promoter.To assay the activity of the promoter, the fragment was amplified by a new pair of primer from the clone vector and constucted a new GUS expression vector called pPGGUS by replace the CaMV 35S promoter pBI12. Agrobacterial mediated transformation of the pPGGUS to tabaco and cherry leaf discs, regenerated plants from tabaco and regenerated root from cherry leaf discs were aquired on Kan suppliment medium. GUS assay showed that GUS is expressed in tobaco leaf and root, and also expressed in cherry root. It implied that we have cloned a fragment including the promoter of PGIP gene. The work of clone is still in process.