Cloning And Functional Analysis Of A PsPGIP Gene From Polygonum Sibiricum Laxm

Polygalacturonase inhibiting protein (PGIP) are primarily localized in the cell wall and endolpasmic system, and they areinhibitors of endo-polygalacturonase enzyme (endo-PG) from fungi, which refrain plant from fungal infection, PGIP have discovered and studied both in monocotyledonous and dicotyledonous plants. The cDNA sequence of the PGIP was cloned, based on PGIP'EST sequence obtained from SSH library of Polygonum sibiricum Laxm.,using RACE technology, and has been carried on the sequence analysis. Real-time PCR show that the expression of PsPGIP gene is differences in leaves, stems and rhizome of Polygonum sibiricum Laxm. under 3% NaHCO3 stress. At the same time PsPGIP expression vectors have been constructed, and the transgenic plants for the PsPGIP gene were generated. untransgenic tobacco and PsPGIP gene over-expression were selected for the test of 3% NaHC03 tolerance. MDA content, SOD activity, POD activity and were measured. As well as in different fungal infection upon by the leaves of the extent of injury analysis. For further breeding for disease resistance and provide a theoretical work experience. The main research as follow:1.The full-length cDNA sequence of the Polygalacturonase inhibiting proteins (PGIP) gene was cloned successfully, the gene is named as PsPGIP(ACD01043).The cDNA sequence of the PsPGIP is 1251 bp in length, The open reading frame (ORF) is 1020 bp in length, encoding a deduced amino acid sequence of 339 residues, and 24 residence of a conserved leucine-rich fragment. Sequencing analysis revealed that the gene belongs to the PGIPs family gene as it contains an amino-terminal (N-terminal) signal peptide and typical conservative region of PGIPs family. The analysis by Real time-PCR shows that PsPGIP gene was distributed in leaves,stems and rhizomes of Polygonum sibiricum Laxm.. Under inducing expression under 3% NaHCO3,the espression of PsPGIP gene was induced by salt obviously, by which we can conclude that PsPGIP gene palys a very important role in the salt-resistant.2.Genetic transformation of PsPGIP and stresss assay of transgenic plants(1).Construct the expression vectors successfully, the transgenic plants for the PsPGIP gene were generated by an Agrobacterium mediated method. The expression analysis by PCR and RT-PCR showed that PsPGIP was expressed in tobacco.(2).Six transgenic lines were selected for the test of NaCl tolerance. MDA content, SOD activity, POD activity and soluble protein content were measured under this stress. The results showed that SOD activity, POD activity and soluble protein content in most transgenic lines was higher than that in untransgenic tobacco under 100 mmol/L NaCl conditions. Furthmore, MDA contents in most transformed plants were lower than that in untransgenic tobacco under 100 mmol/L NaCl stress. The results demonstrated that transformed plants exhibits salt stress tolerance were higher than that in untransgenic tobacco.(3)Six transgenic lines were selected for the test of fungal infection tolerance.The results showed that compared with untransgenic tobacco, all transgenic lines were up-regulated under the F.oxysporum and anthracnose infection stresss, transgenic lines enhanced the tolerance of fungal infection stress.