Stessman, Dan , Portis, Archie, R .
Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein.
The cDNA , ArRbcS3, for the small subunit of Rubisco from the Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to ensure delivery and processing of the mature protein in the chloroplast. In addition, the expression of the native tobacco rbcS gene family was inhibited by RNAi. Due to the sequence similarity of rbcS genes across different plant species, the RNAi construct was targeted to the 3’ UTR of the tobacco rbcS to ensure noninterference with the expression of the ArRbcS3. Northern blot analysis of transgenic plants revealed a strong inhibition of tobacco rbcS expression while ArRbcS3 was strongly expressed, indicating that the RNAi was specific for the tobacco genes but did not interfere with the expression of the foreign rbcS. Western blots of 1D and 2D protein SDS-PAGE gels also revealed a greatly reduced, but not a total loss, ofÂ native RBCS protein. 2D analysis showed that the foreign expressed ArRBCS migrated to the proper size and pI. The transgenic tobacco with both the RNAi and ArRbcS3 constructs were able to grow under ambient CO2 concentrations, although somewhat impaired, whereas the RNAi only plants required elevated CO2 to achieve sufficient growth to maturity. These results give some indication that a hybrid Rubisco with tobacco large subunits and Amaranthus small subunits is formed and is partially functional.
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1 - United States Department of Agriculture, Agricultural Research Service, 1201 W. Gregory Dr., 193 ERML, Urbana, IL, 61801, USA
2 - United States Department of Agriculture, Agricultural Research Service
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM