| In order to explore the mechanism of phloem mRNA distant transmission and the possibility of FT mRNA moving in woody plants,This study constructed over-expression vector of 35S::PtFT:GFP、35S:: PtFT:GFP:tRNA22、35S:: PtFT:tRNA22 and ectopic expression in tomato.Wild-type tomato grafted with transgenic tomato.The GFP fluorescence as a tracer to study FT conduction ability,direction,pathway and distribution rules.To investigate whether the tRNA22 engineered FT mRNA structure will enhance the ability of FT to promote flowering of transgenic plants in woody plants,we added a tRNA22 tail after the PtFT and PtFT: GFP gene sequences.The main results are as follows:1.Laboratory preliminary using the transcriptome sequencing analysis shows that a total of 1073 mobile mRNAs were found.61 genes were selected for further verification.Using the PCR product sequencing analysis,We found that there are a total of 15 genes have been moved at the mRNA level,of which 8 genes can be transferred from the scion to rootstocks,5 genes can be transported from the rootstocks to the scions,2 genes Bi-directional movement can occur at the RNA level.2.The TLS-like structure of the detected mRNA was scanned by using the PlaMoM(Plant Mobile Macromolecules)TLS finder online analysis tool(http://www.systembioinfo.org/plamom/)It was found that 8.6% and 14.9% of up-shifted and down-shifted mRNAs respectively have hairpin structure,suggesting that genes with tRNA-like structures are more likely to contribute to the stability of mRNA movement or migration.3.Expression vector of 35S::PtFT:GFP 、 35S::PtFT:GFP:tRNA22 and35S::PtFT:tRNA22 were constructed and transformed into tomato respectively.Transgenic lines were obtained and then positive detection from the level of DNA and transcriptome level respectively were also performed.4.The phenotypic analysis of transgenic lines.The results showed that,PtFT:GFP,PtFT:tRNA22 overexpression of tomato showed earlier flowering compared with wild type tomato.PtFT:GFP:tRNA22 overexpression of tomato did not appear obvious earlier flowering.The results showed that overexpression PtFT:GFP and PtFT:tRNA22 both can promote the early flowering of tomato.5.The wild-type tobacco was grafted onto the overexpression PtFT:GFP:tRNA22,PtFT:tRNA22 transgenic tomato respectively.Using fluorescence microscopy studied FT protein moving.In the wild-type scions,the fluorescence signal was detected at 1 cm of the grafting union,and the fluorescence signal disappeared at 4 cm of the grafting union,indicating that FT:GFP:tRNA22 gene can move at the protein level.However,no exogenous PtFT mRNA was detected at the scions,indicating that the mRNA of exogenous PtFT:GFP:tRNA22 and PtFT:tRNA22 failed to move between the rootstocks. |
