Cloning And Functional Analysis Of Systemic Acquired Resistance Related Gene OsSRT2 In Rice

People have found a number of R gene particularly by genetic engineering. Disease resistance (R) genes protect plants from fungal, bacterial, viral, and nematode pathogens as a complex genetic defense system. Numerous R genes have been cloned and characterized from dicotyledons. Disease resistance (R) genes in plants encode products that specifically recognise incompatible pathogens and trigger a cascade of events leading to disease resistance in the host plant. Biosynthesis of SA in response to pathogens is believed to be controlled by PAD4, EDS5 and SID2. Pathogen-induced expression of PAD4, EDS5 and S1D2 was suppressed by AtSRT2, suggesting that AtSRT2 plays an important role in regulating SA synthesis. Since Sir2 was first isolated in yeast, its homologs have been cloned in mammalian cells successively. Nevertheless, the features of Sir2 proteins are not totally comprehended in plants.In order to detect the role Sir2 played in the resistance progress of rice, we detected where the gene is located, the relationship of expression levels of OsSRT2 and the disease-resistance related levels of SA in transgenic plants, how strong the transgenic plants resist to Xoo pathogen. The details are as the following:1. First, OsSRT2 gene is obtained, electrophoretic shows a sign of 938bp, identical with the forecasted size of the gene, and no phenomena of towing and multi-band dispersions. After sequencing, it was submitted to NCBI Blast website. There is a sequence homology of 99% with the registered SRT2 gene on NCBI, with which there is 100% protein homology. Login the Expansy website and enter protein sequence of OsSRT2, it was deduced that OsSRT2 gene encode a protein, of which molecular weight Mw= 34.56kD, isoelectric point pI= 8.76, which is coorderation with the protein expression in protokaryon. Thus, synonymous mutations was occurred in the nucleotide of SRT2.2. In order to find the fuctional location of OsSRT2 and lay the foundation of speculation of the action mode, gene gun was used to transforme 35S-OsSRT2-G fusion expression vector into onion epidermal cells. Then, subcellular localization of 35S-OsSRT2-GFP was detected by fluorescence microscopy. The result showed that OsSRT2 instantaneous located in the nucleus.3.Via agrobacterium-mediated conversion technology, they were introduced into the rice.9 T] generation over-expressing positive plants,8 plants of T1 interference positive plants were gotten, In vivo generation of free state salicylic acid (SA) levels were measured in T1 transgenic plants by HPLC. In vivo levels of salicylic acid in OsSRT2 over-expression plants were tremendously lower than the control; while the content in OsSRT2RNAi Plants Free SA is about 1.5 times than the control. Descripting OsSRT2 is involved in regulating SA accumulation in the body of the plant, inhibiting SA content in the plants, and thus indirectly demonstrating antiviral signaling processes involved in SA-mediated OsSRT2.4. In order to further illustrate the effect of OsSRT2 from the gene level. The OsSRT2-expression patterns were analyzed by Quantitative PCR. OsSRT2 content in over-expression plants was twice higher than that of wild-type. A certain decline was occurred after SA treatment, but a summit was maintained. Content of OsSRT2 in interference plant is about half of the wild type, a certain decline was occurred after SA treatment, a weakened expression was founded.5. OsSRT2 over-expression plants,OsSRT2-RNAi plants and the control Nipponbare in seeding stage and booting stage were incubated by Xoo pathogen separately. The results indicated that the anti-disease ability of transgenic plant OsSRT2-RNAi was obviously improved; however, OsSRT2 over-expression was disease sensitive.The above statements show that cloned OsSRT2 gene is associated with the disease resistance of rice via negative regulation. System acquired resistance (SAR) is an important part of plant resistance systems, and SRT2 gene is the key gene in the SAR system. Therefore, the successful acquisition of OsSRT2 gene and the relative researches not only demongstrate the mode of function of OsSRT2 and the role it played in the resistance progress, but also provide a meaningful purpose gene for the use of genetically engineering of rice resistant varieties, and are of significant values in applications.