Isolation And Expression Of StDIR1and StDIR2Responsive To Infection By Phytophthora Infestans

Potato is one of the most important food and vegetable crops in the world. Late blightcaused by Phytophthora infestans, however, has brought a great loss in the whole world’spotato production; therefore, development of new varieties resistant to late blight bytransgenic technology has become an important project in potato breeding. Dirigent proteinexisted in all the vascular plant plays an important role in plant disease resistance, as it isrelated to the plant’s lignin biosynthesis which acts as the natural barrier against the infectionof pathogen to the plant cell. Specific primers were designed according to the potato ESTsequences, and two full-length cDNA sequences designated StDIR1and StDIR2were clonedthrough the RACE method. The expression analysis of these two genes under the treatmentwith P. infestans, exogenous H₂O₂and NO, as well as the tissue-specific expression analysisof StDIR1were discussed in this study. The results are shown below:（1） The full-length StDIR1cDNA sequence （729bp）, which encoded a polypeptidecomposed of191amino acids, was cloned. The putative polypeptide contains a signal peptideof24amino acids. Theoretical molecular weight and isoelectric point of the protein is21,315Da and9.37respectively, and it shares a similarity/identity of76%/62%with Gossypiumhirsutum dirigent-like protein1.The full-length cDNA seqence （778bp）, designated StDIR2, encodes a polypeptidecomposed of194amino acids with a signal peptide of26residues. Molecular weight andisoelectric point of the protein is20,929Da and9.12respectively, and it shares asimilarity/identity of74%/58%with Picea glauca dirigent-like protein pDIR12.Bioinformatic analysis indicated that StDIR1and StDIR2, which contain differentN-glycosylation sites, are secretary proteins belonging to DIR-b subfamily.（2） The expression of StDIR1is up-regulated in different degrees under the induction ofP. infestans, exogenous H₂O₂and NO, respectively. The maximum expression of the gene inlate blight susceptible varieties is significantly later than resistance ones.StDIR2gene can also be induced under the infection of P. infestans, exogenous H₂O₂and NO. The level of StDIR2transcrip reaches the first peak at the induction of6h and the valleyat24h. After the infection of24h, the expression is up-regulated again and remains a highexpression level.（3） StDIR1gene can be induced under the infection of injury, and has the highestexpression level under the infection sometime between24h and48h, while under thecombined induction of injury and P. infestans, the highest expression level12h. But theinduction of StDIR2has no simple trend.（4） Tissue-specific expression analysis indicated that StDIR1is preferentially expressedin roots, with a gradually decreased level in stem, leaf, and flower. But in the tuber tissue,StDIR1has a relatively high expression level in the resistant varieties, while almost noexpression was detected in susceptible plants.