Rice Genetics IV - IRRI books - International Rice Research Institute

Table 3. The levels of green fluorescent protein (FLU, fluorescent units, per microgram protein)in transgenic and nontransformed rice plants.GFP plasmid Number of R 0lines Range of Average GFP level Variation inGFP values (fold difference) a transgene expressionpSBG700 12 16.8–84.1 47.1 b (1.0) 5.0-foldpJPM5/MAR 10 605–1,333 867 c (18.4) 2.2-foldpJPM9/MAR 6 66.8–356 155 d (3.3) 5.3-foldNone 6 4.4–8.0 5.8aFold difference is the difference in green fluorescent protein (GFP) level between plasmids with thematrix attachment region (MAR) sequence compared with those without the MAR sequence (pSBG700,with the value set at 1.0-fold). The values were not corrected for the background in the nontransformedcontrol. b Standard error is 3.2; standard deviation is 12.5. c Standard error is 65.8; standard deviationis 132.2. d Standard error is 12.6; standard deviation is 54.6.To study the effects of increased distance between the Rb7 MAR sequence andthe actin1 promoter, we used plasmid pJPM9, in which a 2.3-kb HindIII fragmentfrom lambda DNA was inserted between the MAR sequence and the actin1 promoter.The rice actin1 promoter drives the GFP reporter gene expression in pJPM9. Weselected this fragment after confirming that there was no homologous sequence correspondingto the 2.3-kb HindIII fragment in the rice genome by DNA-blot analysis.The average GFP value in six R 0 transgenic plants transformed with pJPM9 was 155FLU µg –1 protein (Table 3), which was only 3.3-fold higher than that of the pSBG700control plasmid without the MAR sequence. The average GFP values in pJPM9 plantswere only 20% as high as those of pJPM5-transformed plants. Moreover, the GFPvalues in the different transgenic plants transformed with pJPM9 varied up to 5.3-fold, which is more than the 2.2-fold variation in plants transformed with pJPM5.We have shown that the tobacco Rb7 MAR sequence significantly increased theaverage GFP values in leaves of transformed rice plants. To test whether the Rb7MAR sequence also acts as an enhancer element in rice, we conducted transient assaysusing 14-d-old calli derived from mature rice embryos. Plasmids pJPM5 andpJPM9, with the Rb7 MAR sequence flanking the gfp-bar cassette, were introducedinto calli by the biolistic method. We used plasmid pSBG700 (without the MAR sequence)as a control. Nonbombarded calli were used as an internal control as a referencewhile quantifying GFP expression of the bombarded calli. GFP expression wasvisualized in calli under a BH-2 Olympus Fluorescence Microscope (data not shown).The GFP transient expression values were measured 48 h after particle bombardment.The average relative GFP value in the pSBG700 control plasmid was 364 FLU µg –1protein, whereas the values for pJPM5 and pJPM9 were 620 and 375 FLU µg –1 protein,respectively (data not shown). Thus, from these experiments, the tobacco Rb7MAR sequence increased the GFP value by only 70%. This minimal stimulation canbe attributed to an enhancer effect rather than an effect on the chromatin structure. Itappears that, in order for the Rb7 MAR sequence to greatly enhance transgene expressionin our system, integration of the plasmid into a chromosome is required,suggesting that this is mainly due to a boundary effect.Transgenic approaches for generating rice . . . 431