Screening Of Agrobacterium Tumefaciens Responsive Gene In Wheat And Research Of The Function Of TaUGT In The Process Of Interaction Between Wheat And Agrobacterium

Wheat is one of most important food crops worldwide, which yield and qulity affects the foodsecurity directly. Genetically modified (GM) technology is of great significance to wheat geneticalimprovement. A common limitation in wheat biotechnological breeding is the low transformationefficiency. Remarkably, gene gun-mediated transformation in wheat often triggers many seriousproblems like high degree of chimeric, multiple integration sites for foreign genes, and transgenesilencing. Howerver, Agrobacterium-mediated transformation can effectively avoid or alleviate thesedrawbacks. Still, Agrobacterium-mediated transformation efficiency in wheat needs to be improved.This study intends to screen and characterize the genes responsible for the transformation efficiencyfrom the view of molecular mechanisms underlying the interactions between wheat andAgrobacterium cells during their co-cultivation. The main results achieved in this study were asfollows:1. According to the results of suppression subtractive hybridization (SSH), proteomics andRNA-seq,20,90, and4,889differential expressed genes were got in response to the Agrobacteriuminfection in wheat, respectively. Of the three methods above, RNA-seq is the most effective, accurate,and suitable for screening differential transcripts on the whole. Based on clustering analysis, most ofthe differential transcripts were involved in metabolic pathways; the second is in secondarymetabolism. Of those,3%participated in plant-pathogenic interaction pathway known so far.However, few were found in biotin synthesis pathway, arachidonic acid metabolism pathway, andphotosynthesis pathway.2. KEGG pathway analysis indicated that wheat UDP-glucosyltransferase encoded gene (TaUGT)was mainly involved in ascorbic acid metabolic pathway and drug metabolism-cytochrome P450pathway. And these two pathways were related to the process of plant disease resistance. qRT-PCRshowed the expression of TaUGT upregulated at12h after Agrobacterium infection and decreasedgradually following the time span of co-culture in wheat. It inferred that TaUGT played a regulatingfactor in the initial stage of infection by Agrobacterium.3. Structural analysis revealed that TaUGT in wheat was representative of those nuclear encodedwithout intron available in eukaryote, which showed high identity with its wild relative-orginatedspecies in evolution. Inferiring that there are nine copies were present in wheat genome based onSouthern blot analysis and the wheat mapped expression sequence tags (EST) in GrainGenes database.And each three copies are located on genome A, B, and D of wheat, respectively. Furthermore,according to the result of subcellular localization, TaUGT located on cytomembrane and function wellthrough transmembrane.4. Functional identification of TaUGT showed that overexpression of TaUGT inhibited transientexpression of GUS in Arabidopsis and wheat after Agrobacterium infection, reduced theAgrobacterium population attaching to the surface of wheat cells, and decreased the Agrobacterium mediated transformation efficiency in tobacco. On ther other hand, inhibition of TaUGT expressioncan increase the amount of regeneration seedlings but not the transformation efficiency in theAgrobacterium mediated transformation using wheat mature embryo derived callus as target tissues.Besides, in vitro eukaryotic expression of TaUGT had no effect on the growth of Agrobacterium.In conclusion, many genes involved in wheat normal metabolism pathway all showed highrelevance to the resistance to Agrobacterium infection in wheat. TaUGT played a defensive role in theprocess of Agrobacterium infection before T-DNA integrating into genomic DNA of wheat.