| Potato(Solanum tuberosum L.) ranks as the world’s fourth largest food crop with a good upside for yield, which will play an important role in national food security. Potato can be attacked by lots of pathogens and insects in the production process, including the pathogenic Phytophthora(Phytophthora infestans (Mont.) de Bary), late blight, which causes a devastating loss of yield. The potato fight against late blight disease through PTI or ETI process will produce the reactive oxygen burnst. Previous research on the STBL-SLG/PK’s homologue gene in Nicotiana benthamiana suggested that the ability of the resistant to Phytophthora infestans strain88069was weakened in silenced leaves. The function of the gene remained unrevealed. To study the mechanism of resistance,we will clone the resistance gene StBL-SLG/PK and analyze the function. The main results are as follows:1. Basing the information from the PGSC and NCBI database cloned the target gene in cDNA and gDNA from E3, showed that the StBL-SLG/PK(Solanum tuberosum Bulb-type mannose-specific lectin/S-locus glycoprotein protein kinases) is a novel intronless gene. The full length of the StBL-SLG/PK cDNA is2373bp, deducing790amino acid and predicting a Ser/Thr kinase protein.2. Treated potato plants with SA, MJ, ETH, BABA and ABA, the StBL-SLG/PK was induced by MJ with high abundance from12to48h and ETH from24to48h, but littile by the others. Drought and salt stress treatment lead the expression of StBL-SLG/PK rise from the beginning to the end. That suggested that the StBL-SLG/PK might have function in the JA-mediated pathway, ETH-mediated pathway and Osmosis-mediated pathway.3. Transient expression of StBL-SLG/PK in onion epidermal cells revealed that it localized in the nuclear membrane. To identify the protein function in vitro, StBL-SLG/PK fusion protein was expressed in prokaryotic expression sysyterm and purified for the kinase assay. This protein possessed self-kinase activity when treated by γ32-ATP radioactive autoradiography.4. The StBL-SLG/PK expression vector were constructed and transformed it into potato E3to study the StBL-SLG/PK function in potato, obtained5RNAi transgenic lines and20overexpression transgenic lines. The overexpression lines improved their resistance to late blight while RNAi lines were more susceptible to the pathogen than to control which could be confirmed by inoculating P. infestans on the transgenic leaves, suggesting the StBL-SLG/PK played a positive role in the resistance of potato against P. infestans.5. Silencing the StBL-SLG/PK of homologue gene in N. Benthamiana by VIGS, the resistance to late blight was decreased in silenced leaves. And the resistance to different races of P. infestans varies, showed that P. infestans LJX18is more infectivity than P. infestans88069on the silenced leaves. To prove whether the gene participate in the STO and R3α-mediated HR response pathway (Hypersensitive Response), the STO/IPIO, R3α/Avr3α and INF1were used to do the transient expression. The result showed that the StBL-SLG/PK didn’t take part in this genes mediated HR response pathway. In order to verify while the gene played an important role in basic resistance pathways, the histochemical experiment mighe be provide some proofs for this. The result showed that the reactive oxygen specie is significantly decreased in StBL-SLG/PK transient silenced leaves and the scavenging superoxide anion is significantly increased and callose is accumulated in vaccination. That suggested that StBL-SLG/PK may regulate the activing oxygen scavenging and participate in the basic resistance pathway. |
