Title of Thesis

Cloning And Over-Expression Of A Germin-Like Protein Gene For Its Functional Analysis

Author(s)

TAYYABA YASMIN

Abstract
Germin and germin-like proteins (GLPs) constitute a large and highly diverse family of developmentally regulated proteins showing a wide range of distribution from Myxomycetes to flowering plants. These proteins have been cited in various plant organs like roots, leaves, nectar glands and seeds etc. Germin-like proteins also seem to be involved in many stress related processes, but their definite role in these processes remains obscure. The present study was conducted to elucidate the function of germin-like protein gene (OsRGLP1) of rice by over-expressing OsRGLP1 cDNA under the transcriptional control of strong promoter in tobacco. The cDNA of OsRGLP1, 958 bp was successfully cloned in pTZ57R/T cloning vector. OsRGLP1 cDNA was inserted in p1301 in sense direction down stream of CaMV35S promoter and transformed into Agrobacterium strain EHA101. Transgenic tobacco plants were produced through Agrobacterium mediated
transformation and confirmed through hygromycin resistance and PCR amplification of OsRGLP1. Transgenic plants were assessed for growth, morphology, oxalate oxidase and superoxide dismutase activities. Morphologically no difference was observed between T0 transgenic and control plants except that leaves of some mature transgenic plants showed necrotic lesions. Transgenic plants were evaluated for the presence of H2O2 which is reported to be involved in necrosis and cell death. Interestingly, higher levels of H2O2 were observed in transgenic plants. Seeds of T0 /T1 transgenic plants were similar in shape, size and coat color to the control plants when harvested, whereas, seed coat of transgenic seeds turned lighter in color after one year storage at room temperature. Germination rate and percentage in transgenic T1 seeds was significantly lower than control seeds. Segregation analysis indicated a 1:1 ratio for hygromycin phosphotransferase gene in T1 seeds. There was no difference in fresh/dry weights, plant heights and shoot lengths, number of leaves and nodes of transgenic and control plants. Number of roots in transgenic plants was lower while roots were longer than control plant roots. No oxalate oxidase activity either of native or transgene origin could be observed in transgenic plants revealing that OsRGLP1 may not posses this activity. Significantly higher levels of heat resistant superoxide dismutase activity were observed in transgenic plants as compared with control. This added activity was insensitive to KCN and sensitive to H2O2 suggesting being due to FeSOD activity. Present study indicates for the first time that OsRGLP1 may posses SOD activity along with a striking observation that this activity is FeSOD type instead of MnSOD, if at all observed. Upstream regulatory region of OsRGLP1 was also cloned, sequenced and analyzed to gain insight into the regulatory aspects. The promoter region contained many regulatory elements related to diverse functions, most intriguing were the elements related to biotic and abiotic stress which emphasizes the possible involvement of OsRGLP1 in these processes. These findings may suggest that OsRGLP1 gene product may act as FeSOD responsible for dismutation of stress generated reactive oxygen species into H2O2 which in turn may play important roles like defense against pathogen and root elongation.

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2.1 Germins And Germin-Like Proteins

2.2 Distribution In Plant Kingdom

2.3 Organ/tissue Level Localization

2.4 Cellular/sub-Cellular Localization

2.5 Regulation Of Germins/glps Expression

2.6 Structure And Phylogeny

2.7 Functions

2.8 Role Of Glps In Abiotic And Biotic Stress

3.1 Preparation Of Complementary Dna Of Osrglp1 And Cloning

3.2 Construct Designing
3.3 Agrobacterium Mediated Transformation Of Tobacco
3.4 Regeneration Of Transformants
3.5 Selection Of Transformants
3.6 Characterization Of Transgenic Plants

3.7 Cloning And Sequencing Of Osrglp1 Promoter

4.1 Cloning Of Osrglp1 Cdna

4.2 Recombinant Vector Preparation
4.3 Agrobacterium Mediated Genetic Transformation Of Tobacco
4.4 Confirmation Of Transgenic Plants

4.5 Functional Analysis
4.6 Detection Of Hydrogen Peroxide (H2O2) In Transgenic Plants
4.7 Oxalate Oxidase Activity
4.8 Superoxide Dismutase Activity
4.9 Cloning And Sequencing Of Osrglp1 Promoter

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