Transformation of rice (Oryza sativa L. var IR64) with three rice seed storage gene promoters (GluB-1, GluB-4, and Glb-1) and cloning of rice glutelin promoter GluB-1 into a pCambia-PvFer expression vector

Myla D. Ocite

Date

4-2007

Degree

Bachelor of Science in Biology

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Hazel L. Gaza

Abstract

OCITE, MYLA DADOLE. University or the Philippines at Los Banos. April 2007. Transformation of Rice (Oryza sativa L. var 1R64) with Three Rice Seed Storage Gene Promoters (GluB- GIuB-4 and Glb-1) and Cloning of Rice (Glutelin Promoter GIuB-1 into a pCambia-PvFer Expression Vector

Adviser: Prof Hazel L. Gaza Co-adviser Dr. Philippe Herve

Three promoter-GUS constructs using three different rice seed storage promoters (GluB-1, GIuB-4 and Glb-1) in pCambia expression vector were inserted in 1R64 rice through Agrobacterium-mediated transformation. A total of 317 transgenic rice lines were regenerated, 74 for GluB-1, 112 for GluB-4, 100 for Glb-1 and 101 for the control plasmid Transformation efficiencies were 30.8%, 46.7%, 41.7%, and 19 4%, respectively. Out of 317, 123 transgenic rice lines: 38 for GluB-1, 33 for GIuB-4, 36 for Glb-1, and 16 for the control plasmid, were positive for insertion as revealed through PCR of GUS genes from leaves. Cloning of rice seed storage promoter GluB-1 in pCambia-PvFer expression vector was done to create constructs for rice transformation with directed expression of Phaseolus vulgaris ferritin gene (PvFer) in rice endosperm PvFer and GluB-1 in TOPO vector were digested and each revealed bands corresponding to the inserted DNA fragments and the vector. Alignment of PvFer gene and GluB-1 sequences with published sequences revealed 99% and 100% identity, respectively. GluB-1 was inserted into pCambia-PvFer vector and two constructs were made. One was pC1380-GluB I -PvFer containing Hygromycin phosphotransferase (HPT) gene in the expression vector and the other, pCO380-GluBl-PvFer without HPT gene. Both clones were tested for correct orientation using restriction enzyme digestion and revealed 1.7 kb band, which corresponds to the digested GluB- I promoter in proper orientation and the remaining band, either 10.2 kb or 8 2 kb in size, which matched the rest of the 600 by fragment of the promoter, 0.8 kb PvFer and 6.6 kb pCO380 or 8 6 kb pC1380 expression vectors.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

Thesis

Document Type

Thesis

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