| Potato(Solanum tuberosum L.) is the fourth most important cultivated crop worldwide, and plays a very important role in the industry and agriculture.The United Nations declared the year 2008 as the Intemational Year of the Potato,noting the important role of potato in the global food security.However,the potato is attacked by a wide range of pathogens and insects,including oomycete pathogen Phytophthora infestans,which is the devastating disease of potato.Traditionally,genetic resistance of potato against P. infestans is classified into qualitative and quantitative resistance.Qualitative resistance is mediated by R genes that only provide short-lived resistance in the field as new virulent pathogen races rapidly overcome the resistance controlled by single race-specific resistance genes.Contrarily,quantitative resistance is regulated by many interacting genes that normally does not prevent the infection,but slows down the development of the pathogen at individual infection sites on the plant,and then,the resistance is presumed long-lasting.However,the mechanism of the quantitative resistance to the disease remains to be elucidated,which limit the full use of the resistance resources.In present study,we aim at the molecular mechanisms of resistance through the cloning and foundational analysis of three quantitative resistance-related-genes.The main results are as follows:1.A novel gene,StPK1(Solanum tuberosum protein kinases 1) encoding a protein kinase was isolated by RACE from potato leaves with quantitative resistance to late blight(R1-R11 free).The full length of the cDNA is of 1779 bp,deducing 416 amino acid.The gDNA of StPK1 contains five introns and six exons.The gene was mapped on chromosomeâ…¨using potato diploid population PCC1.The RT-PCR showed that StPK1 was induced by P.infestans rapidly and standing for 36 h.2.A potato gene,StLRPK1(Solanum tuberosum leucine-rich-repeat receptor-like protein kinases 1) was isolated by RACE from potato leaves with quantitative resistance to late blight.The full length of eDNA is 3137 bp,encoding 796 amino acids belonging to leucine-rich repeat receptor-like kinases(LRR-RLKs) with 88%of identity to SRF3 of Arabidopsis thaliana.StLRPK1 contains a signal peptide,five LRR motifs,a transmembrane domain,two proline-rich regions and a serine/threonine protein kinase domain.The transcripts were present at high levels in flowers and young leaves,while low in other tested organs.The rnRNA of StLRPK1 was inducible in potato leaves by P. infestans and showed different profiles when subject to SA,MJ,ETH and ABA.3.A novel gene,Star(Solanurn tuberosum ankyrin-rich repeat) was isolated by RACE from potato leaves with quantitative resistance to late blight.The full length of cDNA is 2047 bp,encoding a peptide with 514 amino acids with 78%of identity to XB3 of Oryza sativa.The deduced amino acids possess five ankyrin repeats and a RING finger domain. The mRNA of Star was mainly expressed in roots,and trace expression was detected in stems and flowers.Transcripts of Star were inducible in potato leaves from 24 h to 72 h after inoculation of P.infestans,and the gene was mainly induced by SA,MJ and ABA from 2 to 36 h,and by ETH from 24 to 36 h.4.RNA interference(RNAi) and sense over-expression mediated by 35S promoter were conducted and the genetic transformations were carded out to further verify if StPK1 could contribute to potato resistance to late blight.There were 22 E3-RNAi(RNAi silencing in cv.E-potato 3),13 E3-pBI(over-expression in cv.E-potato 3) and 7 W-pBI (over-expression in cv.Zhuanxinwu) transgenic lines with 1-3 copies of the gene insertion confirmed by the Southern blot analysis.The transgenic plants were challeged with P.infestans,showing that StPK1 played a negative role in the resistance of potato against P.infestans.5.To approach an understanding how StPK1 acts,we studied the expressions by RT-PCR of 11 potato genes which were documented defense-related and P.infestans inducible in the selected transgenic lines at 24 h after the inoculation of P.infestans.The five genes, StpotLX-3,StrbohD,Stpr1b,StPR-1 and hmg2.2 showed their transcripts influenced remarkably by the expression of StPK1.Supression of StPK1 increased the expression of these genes while overexpression of StPK1 down-regulated the expression of them, revealing that StPK1 was on the upstream of these genes and realized its function in late blight resistance through negative regulation of the defense-related genes in downstream. 6.Treated potato plants with SA,MJ,ETH and ABA,StPK1 was induced by SA with high abundance from 2 to 36 h,but littile by the others.The third compound leaf from the top of 8-weak-old potato plants was induced with SA for 1 h,and the first to sixth compound leaves showed the same expression level of StPK1,indicating SA could be transmited nonpolarly in potato plant and activates StPK1 with a spatio-temporal trigger mode.Furthermore,the five negatively regulated genes were also inducible by SA, implying that,as same as StPK1,these genes may have function in the SA-mediated pathway,and reconfirming that StPK1 could be a coordinator of these defense-related genes in response to late blight attack.7.Mediated by 35S promoter,the tobaccos carrying sense over-expression of StLRPK1 and Star,respectively,were inoculated with P.infestans,and the results showed that transgenic leaves exihibited smaller lesion area than the wild type,suggesting StLRPK1 and Star played a positive role in the resistance of potato against P infestans.
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