Cloning Of PGIP Genes And Their Promoters From Prunus Mume And Prunus Salicina, And Transformation Of Tobacco With PGIP Gene From Prunus Mume

When fungi infect plant, polygalacturonase is the first cell wall degradation enzyme(CWDEs) to be secreted. It can degrade the pectin component of plant cell wall, facilitatingpenetration of plant by fungi. As a result, the plant is ill. Polygalacturonase-inhibitingprotein (PGIP) is a glycoprotein existing in the cell wall of many plants.By recognisingfungal polygalacturonase specifically and controlling.to a certain extent ,PGIP hasprevented fungi from degrading plant cell wall. PGIP may modulate PG activity to preventcomplete degradation of pectin polymers in plant cell wall. Certain breakdown products ofthe plant cell wall are elicitors of plant defense responses. They can make plant's inherentdefense response happen. As a result, PGIP plays an important role during plant controllingfungi invading.Using Prunus mume and Prunus salicina as research materials,we cloned PGIP genesand their promoters and analyzed the structure and function of PGIP genes and theirpromoters by using correlative software. Furthermore, we built a plant binary expressionvector, which can make PGIP gene express efficiently. And by using tobacco as plantmaterial, we studied the transformation and expression on PGIP genes. The main resultswere as follows:1. Using the genomic DNA of leaves from Prunus mume as template, and using theconserved sequences of PGIP gene as primers,we cloned the whole DNA sequence of PGIPgene from Prunus mume by PCR(GenBank locus: AY764131). 2.Using the genomic DNA of leaves from Prunus salicina as template, and using theconserved sequences of PGIP gene as primers, we cloned the whole sequence of PGIP genefrom Prunus salicina by PCR(GenBank locus: DQ200847).3. By analyzing of PGIP gene from Prunus mume and Prunus salicina, we found thatthese two genes are both very homologous to those from Prunus americana, Prunuspersica,and Prunus mahaleb. The identity among them was ranged from 95% to 97%.These two genes both have a complete coding region and typical entron spiced sites. Thededuced protein sequences had ten leucin-rich regions (LRRS), which exists in proteinsresistant to pathogen widely. In the phylogenic tree, they belong to the same cluster as PGIPgene of Prunus armeniaca and Prunus persica and Prunus mahaleb,but was far away thecluster of PGIP gene from genus of Pyracantha and Fragaria and Citrus and Malus. 46nucleotide sites were different between these two DNA sequences, and 15 amino acid siteswere different between these two protein sequences, but none of them occurred in the siteswhere were functional.4. Using the genomic DNA of leaves as template,According to the sequence located inextron of 5'-end of PGIP gene from Prunus mume, we designed a pair of primers in reversedirection, a fragment of 1311bp length was isolated by polymerase chain reaction(GenBank locus: DQ364056). After analyzing the structure of this sequence, we found thatthe exact length of the promoter of PGIP gene was 1038bp.5. Using the genomic DNA of leaves as template According to the sequence located inextron of 5'-end of PGIP gene from Prunus salicina, we designed a pair of primers inreverse direction., a fragment of 2338bp length was isolated by polymerase chain reaction(GenBank locus: DQ364055). After analyzing the structure of this sequence, we found thatthe exact length of the promoter of PGIP gene was 1870bp.6. The BLAST results of Prunus mume and Prunus salicina showed that none ofsequences collected in three main nucleotide databases, GenBank, EMBL and DDBJ, wassimilar to two promoters from Prunus mume and Prunus salicina. By Further analyzing textannotation of one hundred and thirty nine pieces of PGIP in the above three nucleotide databases,we found that none of promoter of PGIP gene existed in the above threenucleotide databases.After analyzing of Arabidopsis and rice genomes,we only found thepromoter information about PGIP gene in genome annotation of Arabidopsis. We exactedthe promoter according to the annotation of PGIP gene from Arabidopsis genome and didthe comparison of promoters among the three promoters. The results showed that there washigh difference between promoter of PGIP from Arabidopsis and two promoters fromPrunus mume and Prunus salicina, However there was only little change between twopromoters from Prunus mume and Prunus salicina.7. The analysis of cis-regulatory elements in promoters from Prunus mume and Prunussalicina showed that, besides the two constituent elements, TATA box and CAAT box, threeelements related to resistance to pathogen, SEBFCONSSTPR10A element, GT1 elementand WBBOXPCWRKY1 element also presented in the two promoters. This indicated thatthe expression of PGIP gene was relative to pathogen invading, wound and Salicylic acidinducing.8. After building a plant binary expression vector with PGIP gene, we transferred it toagrobacterium LBA4404. by agrobacterium-mediated, we transformed tabacco with PGIPgene from Prunus mume, and got twenty-five lines with the resistance to hygromycin.Further detection on thirteen lines among them by polymerase chain reaction showed thattwelve lines had the target strip, Then, we detected seven lines among the twelve lines bysouthern bloting. The results indicated that the PGIP gene had been integrated into theirgenomes. After the further detection of RT-PCR, we found that PGIP gene have expressedin transcription level in all 7 lines. By analysis of cDNA structure,we found that theincision site of entron consisted with our predictive result.