Study On Construction Of Protoplast Isolation, Purification And Regeneration System And Knockout Of Disease Related Genes(NtEIF4E And NtTOM3) In Nicotianatabacum

Plant protoplast is a cell with cell wall removed. Tobacco protoplast is a good tool to study gene function at cell level. It is an ideal experimental material to study plant–virus interaction at the cellular level. What is more, the process of molecular breeding through genetic manipulation would be largely accelerated by regeneration plants from a single protoplast cell.Tobacco is an important model plant and economic plant. Studying on tobacco disease-related genes is helpful to understand plant defense mechanisms and breed disease-resistance plants.. Potato virus Y(PVY) and tobacco mosaic virus(TMV) are two dominant viruses which cause serious economic losses in several kinds of economic plants. Virus relies on host cells to obtain energy and components of replication and translation. Down-regulation of host genes related to virus replication and transportation, such as EIF4 E and TOM3, could effectively inhibit the proliferation of virus.In this study, protoplast isolation, purification and regeneration system have been constructed in cultivated tobacco. In order to obtain a higher yield and better quality protoplast cells, different effect factors on isolation and regeneration of tobacco protoplast have been investigated. On the other hand, Crispr/Cas9 gene editing system has been usedto knockout disease related genes Nt EIF4 Eand Nt TOM3 and plants with targeted mutagenesis have been obtained.1、Isolation and purification of the cultivated tobacco protoplastsProtoplast isolation and purification systems have been constructed in Nicotiana tabacum Honghuadajinyuan.Cellulase enzymes and macerase were used to break down the cell wall. Eight week old seeding were selected to isolate protoplast cells. Tip tissues with veins removed were cut into 1-2cm filaments, then mixed with the enzyme solution, digested for 12 hours in dark conditions at 25℃. Mesophyll tissues were removedat the end of hydrolysis. The purified tobacco protoplasts were obtained after multiple centrifugation.2、Construction of protoplast regeneration system for cultivated tobaccoPlant regeneration from single protoplast cell was achieved in this study. The main impact factors for plant regeneration, PH and mannitol concentration were investigated.Culture medium with p H5.8 and supplement with 0.5M mannitol were found to be the best condition for protoplasts growth and differentiation by comparison. Entire plant could be regenerated from a single protoplast cell after cultivating for about 200 days.3、Protoplast transformation and detection for cultivated tobaccoPEG-mediated transformation was used to transform tobacco protoplasts. PEG4000 produced by Sigma was found to be the most suitable PEG for the Honghuadajinyuan protoplast transformation. Results showed that plasmid with small size had a high transforming efficiency than that with larger size.4、Crispr/Cas9 gene editing vectors construction and mutation detection of tobacco Nt EIF4 E and Nt TOM3Gene editing vectors CRISPR/Cas9-Nt EIF4 E and CRISPR/Cas9-Nt TOM3 were constructed successfully, totally 9 targets. And we detected the positive reaction, and finally got 7 transgenic plants. In CRISPR/Cas9-Nt EIF4 E transgenic plants, we found 3 target sites mutated, target1 showed a adenine redundant, target 2 mainly in the form of single-base mutation, target3 showed a guanine deleted. In CRISPR/Cas9-Nt TOM3 transgenic plants, we only detected a mutated target site, mainly in the form of deleted 1 to 3 bases in front of the PAM, ratio of mutation was about 12.5%.