Functional Validation Of Melatonin Synthesis Relate Gene In The Process Of Wheat Infected By Puccinia Triticina

Wheat leaf rust caused by Puccinia triticina is a serious disease affecting wheat yield and quality,and has the characteristics of strong infection and wide spread.In the incompatible combination,when leaf rust infested wheat mesophyll cells,mesophyll cells in contact with the sputum parental cell(HMC)rapidly died,induced hypersensitive reaction(HR),and the leaf rust fungus It is impossible to continue to absorb nutrients from mesophyll cells,thereby limiting the growth and reproduction of pathogenic bacteria and limiting the disease to a certain extent.Reactive oxygen species(ROS)is one of the manifestations of plants under external environmental stress.The preliminary work in the laboratory showed that the content of H2O2 were closely related to HR during the infection of wheat leaf rust.Melatonin,as an important active oxygen scavenger in plants,can effectively play an important role in plant resistance to stress,and therefore has high research value.Plants can synthesize melatonin by four consecutive enzymatic reactions using tryptophan as a substrate.It is known that Tryptophan decarboxylase(TDC)and N-acetyls-methyltransferase(N-acetylserotonin)Methyltransferase,ASMT)are the first and second rate-limiting enzymes in the melatonin synthesis pathway,respectively.The wheat TDC1 and ASMT1 genes were significantly up-regulated early in the different combination by screening the early transcriptome database of wheat infestation established in our laboratory.In this study,the wheat variety "Lovin 10"(L10)and the wheat leaf rust fungi 165,260(165,260)were combined into affinity and non-affinity,respectively,by enzyme-linked immunosorbent assay(Enzyme)-Linked ImmunoSorbent Assay,ELISA)for the determination of melatonin content,using virus induced gene silencing(VIGS)technology,RNA interference(RNAi)technology and overexpression technology to explore the two genes in leaf rust The effect of bacteria on the HR process induced by wheat and its effect on the change of H2O2 content,and the role of TDC1 and ASMT1 genes in the resistance of wheat to resistance to leaf rust infection induced HR,in order to further elucidate melatonin The relationship between wheat and leaf rust in the process of interaction with active oxygen scavenging lays the foundation.The main results of this study are as follows:1.The assay of ELISA were used to detect melatonin and it's metabolites at different time points after interaction between wheat and leaf rust.The results showed that the content of melatonin and it's metabolites in the affinity combination and the incompatible combination increased significantly and remained at a high level in the early stage of infection,and the melatonin and it's metabolite content in the incompatible combination.Higher than the affinity combination.2.The RT-qPCR technique were used to detect the expression levels of TDC1 and ASMT1 genes at different time points after interaction between wheat and leaf rust.In the unaffinity combination,the TDC1 gene were at 8,16,24,48 h.The expression level were significantly higher than that of the affinity combination;the expression level of ASMT1 gene at 4,16,24,48 and 72 h were significantly higher than that of the affinity combination in the non-affinity combination.The expression characteristics of TDC1 and ASMT1 genes in different combinations were significantly different,indicating that these two genes are involved in the defense response induced by wheat and leaf rust at the transcriptional level.3.Inoculate 260 on VIGS plants of TDC1 and ASMT1 genes,respectively,through Rohringer and DAB.After staining,the results of fluorescence microscopy showed that the content of H2O2 induced by TDC1-VIGS and ASMT1-VIGS around the infection point of leaf rust were higher than that of empty vector transformed plants,and the HR expansion area and the number of sputum mother cells were larger than that of empty vector transformed plants.The content of melatonin and metabolites were lower than that of empty vector plants,indicating that TDC1 and ASMT1 genes are involved in the HR during the interaction between leaf rust and wheat.4.The RNAi and OE expression vectors of the TDC1 gene were constructed and OE transformed plants were obtained.