Rice Genetics IV - IRRI books - International Rice Research Institute

We now show the effects of the tobacco Rb7 MAR sequence on transgene expressionin rice in terms of the accumulation of green fluorescent protein (GFP) by usingthree plasmids. Among these three, two plasmids contain two identical copies of theMAR sequence, which flanks a GFP gene. A control plasmid contains no MAR sequences.Figure 2 shows the structure of the three plasmids. Microprojectile bombardmentwas used to separately introduce these three plasmids into rice calli.Transgenic rice plants were regenerated and gene expression was measured in riceleaf extracts of 2-mo-old transgenic plants. By comparing transgenic plants with thecorresponding control plants, transgenic plants harboring each of the two MAR-containingplasmids resulted in average gene expression levels enhanced by 3.3-fold and18.4-fold, respectively, as shown in Table 3. More specifically, we compared GFP reportergene expression in plasmids with and without the Rb7 MAR sequence to studythe effects of the MAR sequence on transgene expression in rice. In nontransformedplants, the autofluorescence values averaged 5.8 FLU (fluorescent units) µg –1 protein.GFP values in 12 R 0 transgenic plants (pSBG700) ranged from 16.8 to 84.1, with anaverage of 47.1 FLU µg –1 (Table 3). When the GFP gene was flanked by the Rb7MAR sequence (pJPM5), GFP values in 10 R 0 plants ranged from 605 to 1,333, withan average of 867 FLU µg –1 protein, an average that is 18-fold higher than that ofthose plants harboring a similar plasmid but lacking the MAR sequence. It appearedthat there was no obvious relationship between copy number of the transgene andGFP level in rice plants containing the Rb7 MAR sequence (data not shown).pJPM5KpnI Cla1 HindIII, EcoRI Xbal NotIMAR Act1(p) gfp pin3’ 35S(p) bar nos MAR1.0 1.3 0.7 1.0 0.75 0.59 0.28 1.0 kbpJPM9KpnIMARλDNAHindIII, EcoRI XbalAct1(p) gfppin3’35S(p)barnosNotIMAR1.0 2.3 1.3 0.7 1.0 0.75 0.59 0.28 1.0 kbpSBG700HindIII HindIII, EcoRIAct1(p) gfpNotIpin3’35S(p)barnos1.3 0.7 1.0 0.75 0.59 0.28 kbFig. 2. Schematic diagram of plasmids pJPM5, pJPM9, and pSBG700. The pJPM5 and pJPM9plasmids contain the Rb7 MAR sequence, whereas pSBG700 does not. MAR = tobacco Rb7matrix attachment region sequence; Act1(p) = rice actin1 promoter with actin1 first intron; gfp= a modified gene coding a green fluorescent protein; bar = the bacterial phosphinothricinacetyltransferase gene; Pin3’ = 3’ terminator region of potato proteinase inhibitor 2 gene;35S(p) = cauliflower mosaic virus 35S promoter; nos = terminator region of the nos gene;lambda DNA = 2.3-kb HindIII fragment containing lambda phage sequence from 25,100 bp to27,479 bp isolated from HindIII-digested lambda DNA.430 Cheng et al