Transcription Of GFP In White Clover Bioreactor System

Plant system has been regarded as a viable alternative for production of high value proteins.Whilst a significant amount of evidence suggests that plant 'bioreactor' can produceheterologous proteins. There is still a need to understand how best to harness the full potentialof plant system. To achieve consistently high levels of protein accumulation it was necessaryfor us to develop our understanding of the relationship between transcription, transcriptstability, RNA structure etc and translation efficiency, efficacy and effectiveness. Stabletransgenic plants, whilst the ultimate goal, are far from a model system and require extensivecharacterization. Thus we are proposing to develop an in vitro model system using plant cellcultures as a tool to further our understanding of heterologous protein accumulation becausethis information can direct and improve our expression strategy when creating stablytransgenic plants as bioreactors. In this project it was of importance that the ability to accurately determine quantitativelythe amount of transcript produced in plant cells, so we were looking at ways to improve thequality, quantity and reliability of the first strand cDNA synthesis. It is well known that thesynthesis of first strand cDNA is an important step when attempting to quantify transcriptlevels. The quantity and quality of reverse transcribed cDNA directly influence the accuracyof the quantification. Using a plant derived α-tubulin as a model system the effect of oligotype and DTT on the quality and quantity of first strand cDNA synthesis was assessed via acombination of semi quantitative PCR and Real time PCR Results indicated that anchored oligo dT significantly improved the quantity and qualityof α-tubulin ?cDNA compared to the conventional oligo dT B26, futher more the amount ofoligo used also had a significant bearing. Similarly omitting DTT from the first strand cDNAsynthesis also enhanced the levels of transcript detected. This is the first time a comparativeanalysis has been undertaken for a plant system and shows conclusively that small changes tocurrent protocols can have very significant impact on transcript analysis. To develop our model system, a combination of agro-infiltration and uniform cell culturelines has been used for the characterization of heterologous gene expression. Using thissystem extended periods of transient transcription could be achieved allowing quantitativeanalysis of transcription. For building the model system to analysis the transcription of GFP, two basic expressioncassettes including double35S-GFP and -90-GFP were constructed. A very strong translationstart site (TSS) was also introduced before the ORF of the GFP gene: the -3 is A and the +4 isG based on the common motif of Kozak consensus, -10 A was specific for improving thetranslationg efficiency of pea and white clover. Two new sites of ClaI and XhoI were alsointroduced into the 5' end of the cassettes, which would be available to insert 5'UTR andother functional motifs. The two basic expression constructs were transferred in AgrobateriaAGL1-pSoup strain as AGL1pSoup-pG0020 and AGL1pSoup-pG0023, which were utilizedto do transient transforming white clover leaves at different developing stages viavacuum-infiltration. Key parameters of the model system have been successfully optimized. The optimizationresults showed that (1) When using Tubulin#2 plasmid as the exogenous control in Real TimePCR, the template concentration for standard curve should be more than 10 ag/ul (2.14 copy).(2) The primer set-BADF159 (300 nM) and BADR160 (300 nM) were used in the followingexperiments. (3) The OD600 value 2.205 of AGL1pSoup-pG0020 was chosen as the parameterfor vacuum-infiltration experiments in plant cells of white clover. The data analysis of the transcripts of GFP in plant cells presented that (1) There wassimilar trend in the amount of GFP for both Geno 4 and Geno 10. The mature leaves producedmore GFP than that of the younger or older ones. For Geno 4, No. 9 and 11 produced moreGFP. But the younger leaves such as No.