INVESTIGATION OF GLYOXALASES IN Brassica spp UNDER ABIOTIC STRESS AND PURIFICATION OF GLYOXALASE-I

A Thesis Submitted to the faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka, in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN BIOTECHNOLOGY SEMESTER: JANUARY-JUNE, 2014

This study was carried out in the Molecular Breeding Laboratory of Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur during the period from March, 2014 to March, 2015 to investigate the role of Glyoxalase system in contrast plants of Brassica spp. Ten prescreened genotypes were subjected to test the variation among them with 5 known SSR markers, and among them, E5 (Tori 7) and E6 (BARI sarisha 16) were selected based on their variation. The comparative phenotyping study of E5 and E6 under salinity and drought stress revealed that E6 is more tolerant than E5 in both stresses. To investigate the responses of Glyoxalase-I (Gly-I) and Glyoxalase-II (Gly-II) in detoxification of methylglyoxal (MG) under salinity and drought stress, 5 day old Brassica seedlings were subjected to impose NaCl-induced salinity level (16dS/m) and drought stress and data were observed at 2 day, 4 day and 6 day of stress. The contents of (MG) increased 2.0, 2.5 and 3.0 fold over control under salinity at 2, 4 and 6 day, respectively in E6 and 0.5, 0.3 and 0.6 fold in E5. On the other hand, in drought stress, methylglyoxal (MG) level increased 2, 3 and 3.5 fold over control at 2, 4 and 6 day, respectively in E6 and 2, 2.5 and 3 fold in E5. Under salinity stress, Gly-I activity increased by 41%, 69% and 105% in E6, at 2, 4 and 6 day, respectively, whereas 12%, 43% and 41% in E5. On the contrary, in drought stress, Gly-I activity increased by 70%, 76% and 61% over control at 2, 4 and 6 day, respectively in E6, and 37%, 32% and 53% in E5. In E6, Gly-II activity increased 144%, 128% and 101% at 2, 4 and 6 day of salinity stress, respectively in E6, and 52%, 59% and 50% in E5. Notably, concomitant increased activities of Gly-I and Gly-II with increased MG suggested their MG detoxification role in Brassica Spp. At the same time, it was remarkable that both Gly-I and Gly-II activities were higher in E6, and hence, Gly-I was purified from E6 seedlings. In purification, Gly-I were found to be highly purified and migrated as a single band on SDS-PAGE with an apparent molecular mass of 27 kDa in silver staining and coumassie brilliant blue staining with purification fold of 113 and recovery of 0.38%. iii