Eliminate Homologous Co-suppression In Plant Transgenesis By Novel Transgenic Vectors

Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Transgenes can also cause the silencing of endogenous plant genes if they are sufficiently homologous, a phenomenon known as homologous co-suppression. Homologous co-suppression occurs transcriptionally and post-transcriptionally but predominantly post-transcriptionally. A major reason for transgene silencing and homologous co-suppression is that aberrant RNA produced by highly transcribed transgenes triggers the RNA interfering machinery of plant cells.Commonly, these transgenes contain no untranslated regions (UTR), no introns and any other intergenic non-coding sequences, which are also prime targets for transgene silencing. To investigate this, we conduct several vectors containing5’UTR、intro、3’UTR and intergenic non-coding sequences’genes respectively in Arabidopsis thaliana and choose Cobra gene as the object to carry out related research. Cobra occures100%homologous co-suppression in a controlled case, while we find the following results by using improved transgenic vector. Firstly, introducing an autologous intron into5’UTR of transgenic Cobra reduces silencing by30%while5’UTR does not preclude silencing. Secondly, placing bicoid3’UTR in the transgenic3’UTR and expressing its binding protein Stau can decrease homologous co-suppression by68%while adding6/12/24ms2and expressing its binding protein MS2is also able to inhibit homologous co-suppression to some extent. Thirdly, when we insert6/12/24ms2affter terminator and express MS2. the occurrence rate of homologous co-suppression will drop by83%. Here, we discuss tentatively towards the mechanism of above results by using the real time quantitative PCR. sequencing site of transcription termination site and some other methods of these results was discussed. Here, we also use fluorescence real-time quantitative PCR and sequencing transcriptional termination sites to conduct a preliminary study on the mechanism of above results. All results show that the transcriptional read-through is common towards exogenous genes, and it is not sufficient to trigger homologous co-suppression. In addition, we also believe that the post-transcriptional gene silencing is the main result for homologous co-suppression.