Study On The Effect Of Wheat Glutaredoxin TaGRXC14 Gene In The Interaction Between Wheat And Leaf Rust

Glutaredoxin(GRX)is a small molecule protein that is widely present in various organisms.Using the pathway enrichment software mapman to screen the differentially expressed genes involved in the oxygen metabolism process in the transcriptome database of wheat-leaf rust interaction obtained in the early stage of our laboratory,and obtain a GRX gene named TaGRXC14(Triticum aestivum Glutaredoxin C14,GenBank gene accession number:MW965452).After PCR amplification,the full length of the gene fragment was 312 bp,and the molecular weight of the encoded protein was about 11.15 kD.The expression pattern of TaGRXC14 gene after inoculation of Puccinia triticina race 260 in the near-isogeneic line TcLr26 of wheat was analyzed by real-time quantitative PCR(RT-qPCR)technology.The results showed that the gene was in the inoculated leaves.There is high expression after rust fungus,and the expression level has two peaks,which shows that the TaGRXC14 gene is involved in the process of TcLr26 resisting the infection of leaf rust fungus.In the prediction of TaGRXC14 protein using bioinformatics methods,the results show that TaGRXC14 is located in the cytoplasm and its protein sequence is highly homologous and conserved(>90%),and it is relatively distantly related to species such as oil palm and small fruit wild banana.Use VIGS technology to silence some fragments of the gene,and then use ELISA and other methods to determine the content of TaGRXC14 protein,and observe and determine the trend of changes in H2O2 content,and then observe HR,etc.with the help of a fluorescence microscope,and explore its presence in wheat and leaves.The function of rust fungus interaction system.This experiment explored the role of TaGRXC14 gene in the process of wheat resistance to leaf rust infection,and proved that TaGRXC14 gene can eliminate excessive H2O2 in cells and further grow.The main results obtained in this experiment are as follows:1.The full length of TaGRXC14 gene is obtained by PCR amplification,which is 312 bp and encodes 103 aa.2.Through bioinformatics analysis,it is found that this gene is a typical CC type GRX,and has its typical CCMC conserved structural sites,as well as important structural domains such as GSH binding sites.In addition,the tertiary structure of its protein sequence was also predicted.By analyzing the homology of its protein and constructing a phylogenetic tree,it was found that it is more closely related to species such as Aegilops tauschii,Hordeum vulgare,Triticum dicoccoides,Brachypodium dislachyon,etc.,It is relatively distantly related to Elaeis guineensis and Musa acuminata.3.Exploring the expression pattern of the gene at the transcriptional level,the results show that in the incompatible combination,the expression level has two peaks at 16 h(that is,the early stage of this test)and 48 h(that is,the later stage of this test)after inoculation with leaf rust fungus.And the peak value at 48 h was relatively higher;and in the affinity combination,the expression of TaGRXC14 gene showed an increase first and then a decrease.The results indicate that the TaGRXC14 gene may play a positive regulatory role in the process of wheat resistance to leaf rust infection.4.The subcellular localization vector of TaGRXC14 protein was constructed.With GFP empty as a control,the leaves were observed using a fluorescence microscope after tobacco injection.The results showed that TaGRXC14 protein was localized in the cytoplasm.5.After silencing the TaGRXC14 gene with VIGS technology,the protein content of TaGRXC14 at different time points of wheat inoculation with Puccinia triticina was determined,and the accumulation of H2O2 was observed by DAB staining.At the same time,the H2O2 content was quantitatively determined,and the development of leaf rust fungus and the expansion of HR were observed.The results showed that compared with unsilenced plants,the expression of TaGRXC14 protein decreased,DAB staining area increased,H2O2 content increased,HR area increased,and the number of HMC increased.It is speculated that after silencing the TaGRXC14 gene,H2O2 excessively accumulates in plant cells,which expands the HR area.This indicates that TaGRXC14 gene negatively regulates HR by eliminating H2O2.6.Constructed and obtained RNAi vector and overexpression vector.