ed with Rhizoctonia solani. There were varied zones of inhibition in the test fungus depending on the protein concentration applied. A graph was plotted between percentage growth inhibition and protein concentration and the IC50 value was calculated to be 38 g/ mL for this pathogen. To observe whether AdTLP exhibits any -1, 3 glucanase activity, 50 g of recombinant protein was incubated with Laminarin for 30 min to 3 d. No detectable absorbance was observed at 595nm indicating that AdTLP did not possess any identifiable glucanase activity. In vitro antifungal activity and endo -1, 3 Glucanase assay Antifungal activity of AdTLP protein was investigated using spore germination and plate assays. During spore germination assays with F. oxysporum, F. solani and 14981513 B. cinerea, drastic Genomic DNA PCR and RT-PCR analysis on putative Y27632 dihydrochloride price transgenic plants To identify the primary transgenic plants with high and low level expression of the target gene, AdTLP, RT-PCR analysis was performed. Genomic DNA was isolated from ten different 6 A Thaumatin like Protein Gene of Wild Peanut 
kanamycin positive plants and PCR reaction was performed by using primers for the marker genes, nptII marker and AdTLP. Out of ten plants analyzed, seven plants gave expected amplification of 700 bp and 726 bp fragments respectively for nptII and AdTLP sequences. This was followed by RNA isolation and semi-quantitative RT-PCR analysis for determining the expression of AdTLP gene in transgenic plants. Actin amplification served as internal control. This analysis showed that the putative transgenic plants 7 and 4 were conferring highest and lowest level of expression respectively. The primary transgenic plants of 4 and 7 were selfed to obtain seeds for subsequent generations of the plants after reconfirmation using PCR and RT-PCR. Rhizoctonia root rot assay Antifungal resistance in transgenic plants was checked using the broad host range fungal pathogen, Rhizoctonia solani that causes seedling rot disease in several crop plants. T2 generation progeny of the low expression AdTLP plant, 4 and the high expression plant, 7 were tested for fungal resistance along with control non-transformed plants in the assay. After 2 days of treatment using the sclerotia of the pathogen, fungal mycelia grew and covered the complete upper soil layer in the cups. The symptoms of infection started appearing in the wild type after 5 dpi. Fungal infection was severe on the control WT plants compared with the transgenic plantlets. After 10 dpi, WT plants became completely wilted and turned brownish black. Infection symptoms were also prominent in the progeny of the low expression plant 4, but the progeny plants of the high expression transgenic plant 7 were completely healthy. To check and compare the infection at root shoot junction, plants were uprooted from the cups and compared. Root growth of WT was completely retarded and the whole plant turned brownish indicating complete necrosis. The progeny plants of the transgenic 7 did not show any symptoms of infection whereas, some symptoms of wilting, necrosis and browning were observed in root and at root-shoot junction in the progeny of the plant 4. These observations showed that the high expression transgenic plant 7 exhibited enhanced levels of resistance against 3986806 the root rot pathogen R.solani and AdTLP is a good candidate gene for imparting resistance against some fungal pathogens in crop plants. doi: 10.1371/journal.pone.0083963.g003 Salinity and oxi
Posted inUncategorized