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20173
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Subject: Biotechnology and Molecular Biology × End date: 2017 × Start date: 2017 × Type: Thesis ×
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    ThesisItemOpen Access
    Metabolite profiling of in vitro established cultures of Picrorhiza kurroa Royle ex Benth in different growth regime(s)
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Parihar, Durgesh; Gaur, A.K.
    The herbs as medicine are being traditionally in practice from ancient time for healthcare to safeguard humanity. These are being used in Ayurved, Unnani, Siddha and Homeopathy with different kind of healing practices in animals as well as human being with different drug delivery systems based on active ingridients.Picrorhiza kurroa Royle ex Benth, a Scrophulariaceae member, is a reputed and highly studied medicinal herb endemic to alpine and sub alpine belts of the Himalayas. Several monoterpenoid glycosides are present which are known as picroside I, picroside II, picroside III, more recently picroside IV are being studied specially for hepatomodulator.The plant has been overconsumed for its valuable glycosides, hence now it comes under endangered category according to Red Data Book. The depleting strategies of the herb in the wild which requires implementation of conservation and sustainable utilization practices. It is essential to apply biotechnological approaches including tissue culture which in turn will also help for the defined conditions to enhance the production of picrosides in vitro. In the present research work, an attempt had been made to assess the quality/quantity of Picroside content, total protein profiling and study of SOD pattern analyses through use of elicitors and phytoregulators. The suspended leaves regenerated from in vitro Picrorhiza kurroa cultures using leaves as explant(s) from germplasm evaluation facility. Different MS supplemented with 0.75 mg/L TDZ + 0.75 mg/L IBA was used as callusing medium and BAP 0.50 mg/L + Kinetin 0.75 mg/L was used for shoot proliferation.GA3 as a Hormonal elicitor was used as elicitor to recognize some facts about the expression of biogenesis pathway protein and the content of picroside-II. Results of elicitor study revealed that it might be speculated that mevalonate/ non mevalonate pathways are being involved in Picroside(s) biogenesis. GA3 downregulates the biosynthesis of picrosides as it also follows the same biosynthetic pathways. Electrophoresis of protein reveals bands like 84,67,58,37,25 kDa which were observed in all samples taken at different days interval while other bands were seen in 12 and 18 days samples harvested from suspension cultures, which implies that some proteins are produced under the increased concentration of GA3. Achromatic regions on the native gel indicate the activity of SOD. They play important roles throughout plant growth and development in vitro. SOD activity was found more where higher concentration of GA3 than differently treated samples Although the exact mechanism is to be explored. These results indicative of the increased amount of GA3 indicative of the picrosides biosynthetic pathways.
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    ThesisItemOpen Access
    Molecular mechanisms underlying the stress protective effect of zinc sulfide nanoparticles in Brassica juncea
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-08) Shinde, Sagar Sharad; Arora, Sandeep
    Application of nanoscience and technology in agriculture have provided a ray of hope for increasing crop productivity in a sustainable manner. In the current project, efforts were made to find out whether treatment of Brassica juncea, an important oilseed crop, with zinc sulfide nanoparticles can prepare them to withstand the impeding water deficit stress and what are the molecular mechanisms associated with the effect of these nanoparticles. Zinc sulfide nanoparticles, synthesized through chemical reduction method, were characterized through UV-visible spectroscopy. Brassica juncea seedlings were treated with different concentrations of zinc sulfide nanoparticles (0, 5, 15, 25, 50 & 100ppm) and one set of seedlings was subsequently subjected to 2-days of water deficit stress, and the other set was maintained under regular irrigation conditions. It was observed that seedlings pre-treated with 15 & 25ppm of nanoparticles were able to withstand the water deficit stress better than the untreated seedlings. The levels of standard stress marker molecules were significantly lower in the seedlings pre-treated with zinc sulfide nanoparticles, as compared to the untreated seedlings. Further a differential expression response of various antioxidant genes of Halliwell-Asada pathway was recorded in the pre-treated seedlings, indicating that the stress protective effect of zinc sulfide nanoparticles was mediated through the improved redox status of the pre-treated seedlings. An analysis of the expression profile of specific mapk genes indicated that mapk3 and mapk9 play a major role in mediating the effect of zinc sulfide nanoparticles.
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    ThesisItemOpen Access
    Cloning of unique immunogenic region of Brassica mitogen activated protein Kinase 3 (MAPK3) in expression vector & purification of MAPK3 protein
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2017-07) Das, Shubhashis; Pandey, Dinesh
    The productivity of rapeseed and mustard is severely affected by Alternaria blight disease which is caused by the fungal pathogen, Alternaria brassicae. So far there is no source of resistance gainst this disease. It is being thought that the disease resistant varieties of Brassica can be developed provided that molecular mechanism of pathogenesis / defense is delineated. MAP kinase signaling pathways are hypothesized to play important role in defense against Alternaria blight disease of Brassica. MAPK3 has recently been shown to play important role in defense response against necrotrophic fungal pathogens e.g. Botrytis cinerea. It was realized the regulation of MAPK3 protein at translational level during pathogenesis/defense response towards Alternaria blight disease will throw light on it’s role during pathogenesis process. However this objective can be fulfilled if monospecific polyclonal antibodies are available against MAPK3 protein. In order to achieve the above objective , the full length sequence of MAPK3 gene was retrieved from Brassica rapa genome and an immunogenic unique region was selected on the basis of BLAST and analysis of B and T cell epitopes. This unique immunogenic region of Brassica juncea MAPK3 was amplified and cloned in pGEMTeasy cloning vector to perform efficient restriction digestion of insert DNA for directional cloning into pRSETC expression vector. The sequence and correct reading frame of cloned MAPK3 gene was confirmed by sequencing. Then the recombinant pRSETC vector was introduced into C41 strain of E.coli for efficient synthesis of MAPK3 protein. The synthesis of 6X-His-MAPK3 recombinant protein of 20.6 kDa was induced by 0.7 mM IPTG and analyzed through SDS-PAGE. The cloned MAPK3 gene contained B and T cell epitopes which were different from epitopes present in other MAP kinases. This suggested the cloning of unique immunogenic region of MAPK3 in the pRSETC expression vector. In next step, MAPK3 protein can be purified by Nickel affinity chromatography and protein can be injected into rabbit for raising monospecific antibody against it.