| Wheat(Triticum aestivum L.)is the largest planting area and the highest yield of crops in world.It plays an important role in national food security and social stability.With the planting area and the yield of wheat increasing,the weed becoming increasingly prominent problem.Especially the gramineae weed,such as Bromus japonicus Thunb.,Aegilops tauschii Coss.,and Alopecurus aequalis Sobol.which are difficult to control and few efficient and specific herbicides can be selected.The consequence is that these weeds,which had become destructive in wheat fields,drastically diminishing the yield and excellence of wheat.Even wheat where weeds are occurring heavily have no yields.Glufosinate-ammonium is a broad-spectrum and sterilant herbicide,which is widely used to control a variety of annual and perennial weeds.Therefore,it is an urgent problem for researchers to explore and create wheat resources that can resist herbicides,developing the planting mode of"herbicidal+herbicide resistant crops".Realized controlling weed in wheat fields simplify and effectively,ensure wheat safety,and increase the competitiveness of wheat products in the international market.In this study,we used glufosinate-ammonium to treat wheat seedlings.Then throuth transcriptional sequencing technology(RNA-seq)to study the genetic mechanism of wheat resistance to glufosinate-ammonium stress.And mined related genes which wheat resistances herbicide,so as to provide theoretical basis and technical support for screening and creating breeding materials resistant to glufosinate-ammonium,and developing the new planting mode.The results of this study are as follows:1.In order to identify and screen the concentration and time of wheat seedlings treated with glufosinate-ammonium for RNA-seq,different concentrations of glufosinate-ammonium solution were stressed to wheat seedlings for different time,and it was found that under 4 times concentration(4×,6960 g.ai/hm~2)of glufosinate-ammonium solution.The phenotypes of nodes treated after 3 h and 9 h were significantly different.There was no obvious dried and dead leaves,and the plant had good integrity,which was suitable for RNA extraction and RNA-seq.2.After data filtering,comparison and correlation analysis,it is clear that the transcriptome data is stable and reliable,and can be used for subsequent analysis.According to the FPKM,by the standard of|log2(Fold Change)|>1 and P-value<0.05 to screen out differentially expressed genes in all samples.The results showed that the gene was significantly up-regulated and down-regulated after 3 h and 9 h stress with glufosinate-ammonium.Among them,2141 and 8614 DEGs of JN6-3h and JN6-9h were up-regulated,and 239 and 5772 DEGs of JQ11-3h and JQ11-9h were up-regulated.The results indicated that wheat seedlings could feel stress signal at 3 h after receiving glufosinate-ammonium stress,and then activated related signal transduction and regulation mechanism,and showed significant response at 9 h.In addition,JN6 had more DEGs involved in response to glufosinate-ammonium stress than JQ11.3.The enrichment analysis of GO function showed that DEGs were mainly concentrated in MF and BP after 3 h and 9 h of glufosinate-ammonium stress in JN6treatment group.In JQ11 treatment group,DEGs were distributed in MF,BP and CC after3 h of glufosinate-ammonium stress.After 9 h,the DEGs were mainly concentrated in MF,followed by BP and CC.Annotation of GO revealed that there were more genes related to protein kinase and phosphorylase which participating in regulation and metabolism in plant stress response.In CC,more genes were classified into plastid,chloroplast and thylakoid intrinsic component of membrane,which involved in photosynthesis in plants.The DEGs classified into MF in JQ11 were mostly related to the activities of transport,transferase and hydrolase.4.KEGG enrichment analysis showed that 3 h and 9 h DEGs of JN6 and JQ11 were mainly enriched in the KEGG metabolic pathway,and the number of DEGs enriched in the secondary pathway of JN6 increased from 504 to 3593 at 3 h,which were noted in 127tertiary metabolic pathways.The number of DEGs enriched to secondary pathways in JQ11 increased from 73 to 2277 at 3 h,and were annotated to 117 tertiary metabolic pathways.DEGs increased significantly after 9 h stress compared with 3 h stress,and DEGs mainly concentrated in amino acid metabolism,carbohydrate metabolism and lipid metabolism pathways.5.The most important transcription factor of DEGs after 9 h was WRKY,followed by NAC,MYB and b HLH.These results indicate that transcription factors play an important role in plant physiological activities such as abiotic stress response,hormone response,growth and development.6.qRT-PCR was used to verify WRKY family genes and P450 family genes in JN6and JQ11,and showed significant differences in gene expression levels,which was consistent with the results of RNA-Seq sequencing,indicating that the transcriptome results were accurate and reliable.7.Sequence alignment and phylogenetic tree analysis of WRKY genes and P450 genes using bioinformation analysis software.It was found that WRKY genes up-regulated had belonged to classⅡWRKY.The homology of WRKY 28.1,WRKY 28,WRKY 76 and WRKY 72B was more than 91%,and the genetic relationship was close.The result of Cytochrome P450 amino acid sequence alignment and phylogenetic tree analysis showed that P450.72A15 and P450.72A14 belonged to protein 15 and protein 14 in subfamily A of family 72,and belonged to the same clade with P450.709B1,with 100%homology.8.Preliminary mining of differentially expressed genes showed that Traes CS4A02G044000 gene was significantly induced by glufosinate-ammonium stress.Through the analysis of the protein domain of the gene,the encoded protein contained 1b561 domain and 1 DUF568 domain.Screening genes from the family which Traes CS4A02G044000 belongs to,12 family members were found to contain 1 b561domain and 1 DUF568 domain.The b561 domain is an important domain of cytochrome b561(Cyt B561),which is an important factor of electron transport and may play a role in the resistance to glufosinate-ammonium stress.In summary,it was found that JN6 had more DEGs involved in resistance to glufosinate-ammonium stress than JQ11,the resistance of JN6 was more strong than JQ11.GO functional analysis showed that the DEGs annotated into MF and BP in JN6 were mostly related to protein kinases and phosphorylases,while the DEGs annotated into MF in JQ11 were mostly related to transport,transferase and hydrolase.These enzymes were involved in cell regulation and metabolism in plant stress response.The analysis of KEGG showed that DEGs mainly concentrated in amino acid metabolism,carbohydrate metabolism and lipid metabolism pathways.The biogenic analysis of WRKY genes showed that there were more WRKY transcription factors of classⅡ.The gene of Traes CS4A02G044000 contained a b561 domain,which was the structure of cytochrome b561(Cyt B561).Therefore,WRKY and CYP families may play an important role in wheat resistance to glufosinate-ammonium.Therefore,the WRKY and CYP family genes could be studied in later,and the genes resistant to glufosinate-ammonium can be further explored,that could provide theoretical basis for the research of wheat resistance germplasm resources. |
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