Functional Analysis Of Solanum Torvum StPGIP Gene Related To Verticillium Resistance

PGIP (Polygalacturonase-inhibiting protein) is an inhibitor to polygalacturonase secreted by pathogenic fungi. It mainly exists in the cell walls and endomembrane system and its content differs with growth time, varieties and organs. It has been reported that PGIP could suppress or delay the fungi growth by delaying the degrade process of pathogenic fungi cell wall.After the construction of the StPGIP over-expression vector, genetic transformation was carried out on potato and tobacco by Agrobacterium-mediated method. A total of 14 hygromycin B-positive tobacco lines and 16 hygromycin B-positive potato lines were obtained.5 tobacco lines and 6 potato lines were determined to be positive by PCR analysis. Southern blot analysis on 2 PCR positive tobacco lines and 4 PCR positive potato lines indicated that the StPGIP gene has been integrated into the genome of the potato and tobacco transgenic lines.Spatiotemporal expression analysis and induced expression analysis of StPGIP by RT-PCR and qRT-PCR revealed the different expression levels in different transgenic lines, and the expression level was higher in those plants with stronger disease resistance. Being compared with wild-type plants, the transgenic lines showed a higher expression level of StPGIP. Both of the transgenic and wild-type plants via the infection of Verticillium dahliae exhibited a higher level of StPGIP mRNA than the plants without infection. Additionally, the StPGIP expression of the transgenic and wild-type plants reached their highest expression point at the same time though there was a great difference in the later period, which can be confirmed by the research on the protein level of StPGIP of transgenic potato plants thereafter.The measurement of the soluble protein content by the SDS-PAGE electrophoresis and the antibacterial experiment in vitro were performed to analyze the StPGIP translation level. Before the infection of V. dahliae, the transgenic plants showed a higher protein level, compared with the wild-type plants. After the infection, the soluble protein content increased with different incensement in different time point. Moreover, the protein extraction from the transgenic plant showed an intensive inhibition on the growth of V. dahliae. The observation of phenotypes on the transgenic and wild-type plants after the infection of V. dahliae revealed that the transgenic plant exhibited a resistance to the V. dahliae infection within a certain time. At the same time, we found that the transgenic lines 11 was a high-resistant line.In conclusion, the overexpresion of StPGIP increased resistance to V. dahliae in transgenic tobacco as well as in transgenic potato, suggesting an evidence for application of StPGIP in plant Verticillium wilt resistance breeding.