Construction Of A Homologous In Vitro Translation System Of Chloroplast Encoded Proteins

A homologous chloroplast in vitro translation system of chloroplast encoded proteins D₁, cytochrome f (Cytf), cytochrome b₆ (Cytb₆) was established to systematically study the molecular mechanism of chloroplast encoded proteins' assembly and regulatory processes. The results obtained here were summarized as follows:Using Site-Directed PCR cloning strategy and chloroplast DNA template, full length genes of D₁(psbA), cytochrome f (petA), cytochrome b6 (petB) with high fidelity were constructed. We cloned full length petB gene with tobacco psbA 85bp promoter to enhance translation efficiency. To avoid 3' overhangs in the following in vitro transcription, double enzyme sites were introduced into the end of different transmembrane regions (TMs)of D₁ (5TMs) and cytochrome b₆ (4TMs). All these genes were constructed into in vitro transcription vector pBluescript II SK/KS(+/-) and verified by sequencing. These provide research basis for studying when and how the chloroplast encoded proteins insert and assembly into the thylakoid membrane.A stable and specific in vitro transcription system with high productivity was constructed. The risk of RNase contamination was reduced by increased time of protease K incubation, increased vortex frequency and short isolation time. Enzyme sites of 5' overhangs were introduced into the end of each gene fragment to make the possibility of specific transcripts. The in vitro transcription results of 10 genefragments showed that its mRNA production was 2.5-11.6mg/ml (purity oD_260/280 =1.75-1.85) which is suitable for the following in vitro translation as exogenous mRNA resources.Chloroplast stoma supernatant 30,000g extracts (S30)with translation activity were prepared by these pathways: Pea seedlings with high protein synthesis and low nucleic degradation activity were grown in a large scale;the isolation time of S30 extracts preparation was reduced;protease inhibitors were added to avoid proteins degradation;S30 protein concentrations were increased (16-25mg/ml). The in vitro translation results showed that using these S30 fractions, the first transmembrane peptide of Cytb6 with tobacco psbA promoter were translated tested through autoradiography. This verifies the ability of this constructed in vitro translation system to express exogenous genes.