Establishment Of Wheat Suspension Cell Line And Transcriptome Analysis Of Wheat Cell-deoxynivalenol Interaction

Fusadum head blight of Triticum aestivum L.is a devastating disease caused by Fusarium graminearum.It not only leads to production reduction but also causes mycotoxin accumulation in seeds which is a great threat to food safty.Deoxynivalenol is the most common nonselective mycotoxin and is one of the most notorious ones.The toxicological mechanism of deoxynivalenol to plant is not very clear now and strategies on deoxynivalenol detoxification are limited.We established a wheat suspension cell line and its agrobacteria-mediated transformation system,investigated the interaction of the cell line and deoxynivalenol cytologically,constructed DON induced and repressed cDNA banks and analised gene expression of wheat cell after DON treatment,with the aim of knowning the toxicological mechanisms of deoxynivalenol to plant cell and screening a batch of detoxification related genes for further research.By using leaf base segment of wheat cultivar Z9023 for callus induction,we established a homogeneous and rapidly proliferated embryogenic suspension cell line. Cell vitality percentage of the cell line is above 60%by cell counting method after nutrual red dyeing,and precipitated cell volume increased by 4 fold in a 8 day subculture period,A volume of 0.5 mL inoculum is the best for suspension cell proliferation in 20 mL culture system.Cell division ability of the cell line reached its climax at the 5^th day after subculture with mitosis rate at 4.5%.We developed a solid double layer culture system for suspension cell line preserving,which can prolong subculture cycle and increase cell vitality and proliferation ability.Agrobacteria-mediated transformation of wheat suspension cell was carried out by using a plasmid containing GFP as target gene and Bar as selective marker.After two rounds of PPT selection,we got transformed calli which are confirmed by reverse transcribed PCR.Crude deoxynivalenol toxins increased wheat cell vitality and proliferation ability at a concentration below 0.1 mg/L and severely damage cell vitality and growth at above 2 mg/L.Higher toxin concentration can damage cell membrane and lead to cell death directly or indirectly.Pure DON also have an inductive effect and inhibitory effect at 15 and 50 mg/L respectively.Wheat Z9023 suspension cells were treated with pure DON at 50 mg/L and harvested at 4?12?48 h after treatment respectively.RNA were isolated from these samples and suppression subtractive hybridization was carried out to construct DON-induced and inhibited cDNA banks.We screened 118 DON up-regulated sequences and 59 DON down-regulated sequences from 960 and 768 randomLy selected clones respectively by digoxin labled dot blots.The 118 sequences belong to 33 contigs and the 59 sequences belong to 56 contigs.20 of the 33 up-regulated contigs can be annotated by BLAST search and so as for 41 of the 56 down-regulated contigs.According to MIPS function catalogue,24%of the annotated genes are responsible for cell rescue and defense,14%are involved in protein synthesis,8%for cell component synthesis,6% for signal transduction and 6%for transcription.DON-induced genes include multi-drug resistance protein?MRP?,cytochrome P450, glutathione S-transferase?GST?,ankyrin-kinase and others.DON-inhibited genes include ribosomal protein?RP?,eukaryotes initiation factor?eIF?,vacuolar proton ATPase subunit, ADP-ribosylation factor and others.Semi-quantification and real-time PCR quantification study of the DON induced genes verified the inducibility,of the genes with known functions,the highest induction rate reached 679 fold and the average is 80 fold.At the mean time,we can see the expression changes at different time point after DON treatment; most of the genes increase their expression sharply and then decrease slowly,reach their highest expression at 12 h after treatment.