| Wheat(Triticum aestivum L.),as an important cereal crop in the word,provides starch and energy for the diets of humans.The high and stable production of wheat is of great significance for ensuring food security in China.However,the lack of soil mineral nutrition and the occurrence of various diseases will seriously affect the growth and yield of wheat.The deficiency of available phosphate(Pi)in the soil is one of the key factors that restricting the growth and production of wheat,so the study on the function and molecular regulation mechanism of the genes involved in phosphate starvation responses(PSRs)will be helpful to the discovery and utilization of wheat genes related to high phosphorus efficiency.The research on the cross function of target nutrient stress genes in the immune system is also contribute to the study on the mechanism of disease resistance and the creation of new germplasm of wheat.In this study,the transcription factor TaMADS2 that involved in low-Pi stress response in wheat was cloned and performed the bioinformatics analysis.And Arabidopsis(Arabidopsis thaliana L.)heterologously expressing TaMADS2 were used to further analyzed its role in low phosphate stress response and the regulatory pathway.Then,the biological function was analyzed in wheat by creating the plants with TaMADS2 overexpression and TaMADS2-RNAi.Meanwhile,this study also studied the role of TaMADS2 in the response to leaf rust infection of wheat.The main results are as follows:1.Long-term low-Pi treatment were carried out on wheat varieties,Zhengmai9023,Xinong 979,Jimai 17,Bainong 207 and Fielder,under the hydroponic condition.Then the expression of TaMADS2 in root were detected though the RT-q PCR,and the results showed that the expression was significantly down-regulated in roots of different wheat varieties under low-Pi treatment for 7 days.Afterwards,short-term low-Pi treatment(0-24 h)were carried out on Zhengmai 9023,the RT-q PCR detection showed that the expression of TaMADS2 was obviously induced at 3 h after low-Pi treatment and then decreased gradually with the extension of stress time.This suggested that TaMADS2 is significantly induced in the early stage of low-Pi stress in wheat and may play an important function in the upstream of PSRs.2.The full-length coding sequence of TaMADS2 was cloned.TaMADS2 belongs to M?-branch of type I subfamily in MADS-box transcription factor family through protein domain prediction and evolutionary tree analysis,and it is a relatively conservative gene in gramineae crops.TaMADS2 has a special gene structure including five exons and four introns,in which the first intron is very long.Furthermore,the TaMADS2 protein localized in the nucleus though the subcellular localization assay,however,it has no transcriptional activation in yeast cell.This indicated that the gene acted through the form of protein complex.Therefore,the yeast two-hybrid screening was performed using the c DNA library of wheat seedling,and some candidate genes,such as IAA-like and TRX-like,were obtained.Its interaction mechanism still needs to be further confirmed and studied.3.Heterogenously expressed TaMADS2 in Arabidopsis,and then the identified T3plants(SM)with overexpressed TaMADS2 and wild type(WT)plants were treated under low-Pi condition.The results showed that the change trends of SM and WT plants after low-Pi treatment were the same compared to the seedlings grown under normal-Pi condition,that is,a significant reduction of primary root length,evidently increase of the lateral root density,decrease of the biomass and accumulation of anthocyanin,but the varied range of SM plants was lower.Moreover,under normal-Pi condition,SM plants showed that the primary root length significantly reduced,the root tip number and lateral root density significant increased,the fresh weight of shoots and roots showed decrease,the anthocyanin content increased,and the root-shoot ratio of total P content increased.These characteristics normally observed in WT plants under low-Pi condition.It is indicated that TaMADS2 affecting the growth of plants,especially root morphology,though regulating the genes that involved in PSRs.4.The differentially expressed genes(DEGs)of the roots of SM and WT plants under different Pi conditions were analyzed via the RNA-seq technology.The results showed that the overexpression of TaMADS2 can cause the significantly differential expression of genes related to PSRs in the root of SM compared with WT under normal-Pi condition,and the variation trend of expression is consistent with that of wild-type after low-Pi treatment,such as AtZAT6,At SPX1 and At PHT1;8.In addition,the DEGs of SM_+P VS WT_+P also include the genes that involved in another ion transport,redox processes and the plant defense processes.Furthermore,we classified the DEGs of SM_+P VS WT_+P using GO enrichment analysis.Five biological processes of GO were markedly enriched: secondary metabolic process,response to oxidative stress,response to karrikin,phosphate ion transport and inorganic anion transport.Through the verification and analysis of DEGs related to PSRs,it was suggested that TaMADS2 mediated the root structure though regulating the ROS homeostasis by AtZAT6,At GSTU17 and TRX,thus it would affect the response of root to low-Pi stress.5.Wheat plants with over-expression and silencing expression of TaMADS2 were obtained through wheat transformation mediated by Agrobacterium tumefaciens.The phenotypic observation of CM and WT plants under different Pi conditions showed that the change trend of CM and WT plants was the same after low-Pi treatment.Under normal-Pi condition,CM5 plants with higher expression of TaMADS2 showed that the growth was weakened,that is,the plant height,root length and fresh weight were significantly reduced compared with WT,but there was no significant difference between CM10 plants without overexpressing TaMADS2 and WT plants.The results suggested that the change of expression of TaMADS2 in wheat could affect the growth and development of plant.6.The leaves of different wheat varieties were infected with leaf rust,and the expression of TaMADS2 were detected at different infection time points.The results showed that TaMADS2 was significantly inhibited at 48 h after infection.Observation of CM5 leaves infected Puccinia triticina showed that overexpression of TaMADS2 increased wheat susceptibility to leaf rust,while silencing TaMADS2 expression by BSMV-VIGS reduced the susceptibility to leaf rust.This study mainly elucidated the function and regulatory mechanism of wheat MADS-box gene TaMADS2 in the PSRs and demonstrated that the role of TaMADS2 in resistance to leaf rust in wheat.The study could provide new gene resources and theoretical basis for genetic improvement and employ of wheat germplasm with high efficient utilization of phosphorus and the disease resistance. |
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