Cloning And Functional Analysis Of A Small GTP-binding Protein Gene Associated With Late Blight In Potato

With the continuous increase of the population and reduction of arable lands, food security has been a critical issue for all countries. Experts predict that, by2050, the developed countries will also need to reconsider the food supply for themselves. Rice, corn and wheat are currently responsible for the food security, but its potential yield has been limited. Therefore, the potato, the largest non-sereal food crop, will be of great importance to meet the food security chanllege. However, the late blight disease, caused by Phytophthora infestans, remains the restrictive factor of potato production. Breeding of resistant varieties is expected an effective way to control the disease, however, the resistance been used in past decades is easily overcome as a rapid pathogen evolution. In recent years, with development of the agricultural biotechnology, disease-resistant genes have been exploited for approaching a more stable and durable resistance. One of the potato genes associated with late blight resistance was isolated in present research, and its function and possible regulatory mechanism in late blight resistance were studied. The main contents are as follows:1. Combining the RACE method with the PCR technology, a small GTP-binding protein gene which was previously identified up-regulated by P. infestance in potato was cloned from a potato clone of horizontal resistance,386209.10. The cDNA full-length of the gene is of889bp, which contains a612bp open reading frame encoding203amino acids. The deduced amino acids showed98%similarity with the NtRabla gene of Nicotiana tabacum, so the cDNA sequence was designated as StRab and registered in the Genbank as EF091876. The sequence analysis showed that StRab has specific conservative regions of the Rab genes, further confirming it is a member of the Rab gene family.2. To research on the function of StRab in late blight resistance, we introduced StRab into the eukaryotic expression vector pBI121under the CaMV35S promoter. In addition, partial fragment (about500bp) of the gene with opposite direction was inserted into the expression vector pHGRV under CaMV35S promoter through recombination for constructing the interference expression vector. Thirty-two over-expression lines and twenty-nine RNA interference (RNAi) lines were obtained by infecting the microtuber slices of cv. E-potato3with Agrobacterium tumefaciens LBA4404. The Southern hybridization analysis showed that StRab has been integrated into the potato genome with1-5copies. The RT-qPCR analysis showed that the over-expression lines exihibited significant higher StRab expression while the RNAi lines did not suppress the gene expression obviously when compared to the wild type control. The inoculation with P. infestans indicated that over-expression of StRab enhanced the resistance to late blight but the resistance level of the RNAi lines did not change in comparison to the control.3. In addition, we also constructed the pBI121-StRab-gfp fusion expression vector. The subcellular localization analysis of StRab was executed by infecting onion epidermis with A. tumefaciens LBA4404. The result indicated that StRab localizes in plasma membranes, providing useful clues for further research.4. To further reveal possible mechanism of StRab in late blight resistance, the yeast two hybrid assay was executed to identify the downstream effectors of StRab protein. Some of these effectors showing interaction with StRab are reported closely related with plant disease resistance, such as scaffold, ERF transcription factor, protease inhibitor, polyubiquitin, polyphenol oxidase, chitinase, etc. According to the information available so far, the StRab gene may function by two ways:the first could be involved indirectly in the disease resistance since StRab protein was considered as a key regulator of intracellular transport vesicles; and the second could directly play roles in the signal transduction pathways or other resistance-related processes through the interaction with the downstream effectors, which is worth further investigation to understand the regulatory mechanism of the StRab gene in potato late blight resistance.