Rice Genetics IV - IRRI books - International Rice Research Institute

Table 4. Copy number of transgenes including rearranged copies.No. of No. of intact No. of GUSPlasmid Line no. Copy no. hybridization expression rearranged expressionbands a cassettes bandspAc1PG-CAM 5 2 1 2 0 +(Agrobacterium 6 4 1 4 0 + bmethod) 7 2 1 2 0 + b8 1 1 0 1 –9 2 1 2 0 +10 4 1 4 0 + b11 1 1 1 0 + b12 3 2 2 1 + bAverage 2.3 1.1 2.1 0.25pAc1PG-CAM 14 5 5 2 4 +(particle 15 1 1 0 1 –bombardment 16 4 4 1 3 +method) 17 5 3 3 2 +18 5 5 1 4 +19 10 6 5 5 +20 6 6 1 5 +21 2 2 1 1 + b22 2 1 1 1 +23 3 2 0 3 –24 2 3 1 2 +26 4 4 1 3 +Average 4.3 3.5 1.4 2.8aThe total number of hybridization bands when a two-cut-site enzyme was used to digest genomic DNA inSouthern blotting. b High expression level of GUS was observed in the anther, including pollen of theselines.plants. The size and number of the expression cassette showed the copy number ofintact and rearranged copies of the transgene. Our results showed that the averagenumber of rearranged copies of the transgenic expression cassette in transgenic plantswith pAc1PG-CAM obtained by Agrobacterium-mediated transformation was only0.25, much lower than the 2.8 obtained by using the particle bombardment method.Transgene expression and DNA rearrangement of expression cassettes. Histochemicalanalysis of GUS expression was carried out on pAc1PG-CAM-transformed R 0plants obtained by both the Agrobacterium-mediated transformation method and particlebombardment method. GUS expression levels were observed in young root tipsof all plant lines transformed with pAc1PG-CAM by the Agrobacterium-mediatedmethod except for line 8. Even though there was one copy of the gus gene integratedinto the chromosome, there was no intact gus gene expression cassette in this linebecause of DNA rearrangement. GUS expression was observed in young root tips ofall transgenic plant lines transformed with pAc1PG-CAM by the particle bombardmentmethod except in lines 15 and 23. Even though there were three copies of thegus gene in line 23 and one copy in line 15, no histochemical GUS expression wasobserved, presumably because of gene silencing related to DNA rearrangement. Inplasmid pAc1PG-CAM, Act1, which is mainly a pollen-specific promoter with low434 Cheng et al