Function Of Potato Late Blight Resistance Related Receptor-Like Cytoplasmic Kinase Gene StPK1

Late blight, the most devastating disease of potato production, is divided into vertical resistance and horizontal resistance. The former is controlled by dominant resistance (R) genes and have race specialty, which can be easily overcomed by the rapid evolution of new virulent races of P. infestans, and the later is controlled by many interacting genes and expected to be more durable and reliable. Therefore, selecting the horizontal resistance variety against late blight has become the pivotal aim on potato breeding. StPK1, a cytoplasmic receptor-like kinase, was cloned from the horizontal resistance variety of potato aganist late blight by the previous work in our lab. It may have some important function in the singal transduction of late blight resistance. Plant protein kinase is one of the most important kinases which play a very important role in disease resistance and stress tolerance as well as plant growth. The preliminary experiments show that, the StPKl can be induced by P. infestans, but the function remains to be identified with further experiments. At the same time, to ascertain the activity of StPK1is required to further understand the mechanism of the gene. Late blight resistance of the transgenic plants and the expression of the fusion protein in E.coil as well as the protein activity were further analysised in this study. The main results are as follows:1. Over expression and RNAi transgenic plants were slected by Realtime-PCR.It showed that the highest silencing efficiency of the RNAi transgenic plants reached to70%, and the higest expression level of StPKl of overexpression lines was600times than that of control.5overexpressed and4interference lines which were slected from the transgenic lines were subjected to late blight resistance assessment. The results demonstrated that lesion expansion rate of the overexpression plants were significantly lower than that of control (E3), and it was significantly greater in the RNAi transgenic plants. It showed that StPKl involved in the potato late blight resistance.2. GFP-StPKl vectors was constructed and transferred into E. coli. The fusion vector was transfomed into epidermal cells of onions by the particle bombardment method and cultivated in the dark for12h. Laser confocal microscopy displayed that GFP-StPK1mainly located in the plasma,Which implicated that StPKl exercised its function in the intracellular space.3. Bacterial expression vector of pGEX-6p-1-StPK1was constructed and the fusion protein can be induced in Rosetta by IPTG. The fusion protein was purified by GST dialysis pillar in vitro and the protein activty was identified by γ-32P-ATP which was marked by isotope labeling. The results showed that the purified fusion protein can ineteract with ATP and it can autophorylate. So StPK1is a protein kinase. It has lay the foundation to find the substrate of StPK1for phosphorylation and further analysis the resistance mechanism of the gene.