Modification of a rice seed storage protein gene: Expression in Arabidopsis

Glutelin is the major storage protein in rice seeds. It represents 75% of the total protein in rice seed endosperm; however, it is low in the amino acids lysine and tryptophan. A genomic clone of the rice glutelin 1 (Gt1) gene containing a 1.8 Kb promoter wasmodified to increase coding for additional lysine and tryptophan using polymerase chain reaction (PCR). Two separate PCRs were used to amplify a 2.9 Kb and a 1.5 Kb segment of the Gt1 gene, which correspond approximately to the acidic and basic domains of the glutelin protein, respectively. Modification of a 57-base-pair region was incorporated into this gene by substitution of specific nucleotides in the primers used for the PCRs. Constructs also were prepared for expression of this modified gene in rice plants, both constitutively and in a tissue-specific manner. To enhance the expression level of the modified Gt1 gene, an additional 3.3 Kb promoter sequence from the same glutelin gene was added upstream of the 1.8 Kb Gt1 promoter to form a 5.1 Kb Gt1 promoter. In another construct, the rice actin promoter was connected to the modified Gt1 sequence. This construct would enable the modified gene to be expressed in cells of a rice suspension culture. To examine expression of the modified gene in a plant without interference from native rice glutelin genes, a plasmid was constructed using a binary vector pSB11 to carry a modified glutelin cDNA along with the selectable marker gene, bar, and was transferred into Arabidopsis plants by Agrobacterium infiltration. Transformed plants were selected using Basta and confirmed with DNA hybridization. Expression of rice glutelin was further tested by Western blot and immuno-staining. It was found that both genomic and cDNA copies of the glutelin 1 gene were difficult to maintain in E. coli and Agrobacterium cells, presumably because of “leaky expression” of the cloned gene. Three methods were used to overcome this problem: (1) T-vector cloning. (2) a large scale miniprep screening procedure. (3) incorporation of E. coli lac operon system in constructs