Identification Of Key Genes Regulating Wheat Seed Dormancy And Construction Of Regulatory Networks

Seed dormancy is one of the characteristics that plants obtain in the long-term process of system development to adapt to the changes of environment,which can ensure the survival of seeds in adversity and prevent seeds from germination in unsuitable seasons.At the same time,the dormancy characteristics and environmental temperature of seeds are considered as the main internal and external factors that affect the resistance to sprouting.In this study,SN5058,a strong dormant variety,was selected as the research object.The expression patterns of dormant seeds were obtained by transcriptome sequencing.Through the analysis of bioinformatics and molecular experiments,the key genes of breaking seed dormancy at low temperature were identified,and the regulatory network of seed dormancy at low temperature and normal temperature was constructed,which provided important support for the research of regulatory mechanism of seed dormancy and germination of cereal crops and the breeding of wheat resistance to ear germination.The main results are as follows:(1)Through the identification of the germination rate of 205 materials with different dormancy levels at low temperature(5℃)and normal temperature(25℃),two typical low temperature sensitive materials(SN5058 and SN5059)and low temperature insensitive materials(SN4701 and SN5133)were screened.The germination rate of SN5058 is close to100% after low temperature treatment,but close to 0% under normal temperature.In this study,SN5058,a low-temperature sensitive material,was used to study the RNA-seq expression profile of dormant seed embryos under low temperature and normal temperature.(2)By analyzing the transcriptome data of SN5058,it is confirmed that the data can cover the transcriptome in the process of wheat breaking dormancy at low temperature.It is confirmed that the dormancy can be broken after 6 hours of low temperature treatment.It is verified by q RT-PCR that the gene expression in the transcriptome data is reliable.21 coexpression modules were obtained by WGCNA analysis,among which the red module was positively correlated with the breaking of dormancy at low temperature.The key gene Traes CS6D01G357700,which regulates low temperature breaking dormancy,was identified in red module by Cytoscape,and named it Ta SPT.(3)The results of subcellular localization and yeast trans activation showed that Ta SPT gene had no transcriptional activity.Combined with RNA-seq data and the expression of Ta SPT in low temperature insensitive materials,it was found that the gene was up-regulated in the early stage of low temperature treatment to affect seed dormancy.The protein interacting with Ta SPT was screened by yeast two hybrid system,and the gene corresponding to the protein was named Ta UEV.The analysis showed that the protein was ubiquitin binding enzyme,which indicated that Ta SPT might regulate seed dormancy through ubiquitination of protein.In addition,the gene of Ta SPT was edited by CRISPR/Cas9 technology.At present,the homozygous positive plants with stable inheritance have been obtained,and the functional verification is in progress.(4)Based on the analysis of WGCNA,the regulatory network of TaPHS1,the main gene regulating seed dormancy,was constructed.Seven genes regulating TaPHS1 and 14 genes regulated by TaPHS1 were identified.The gene Novel12181 was found to be a transcription factor of b HLH family.It was cloned and analyzed that the gene encodes a 303 AA ALC protein and named as Ta ALC.Combined with the regulatory network and expression data,we know that the gene may regulate seed dormancy through positive regulation of TaPHS1.In addition,the interaction proteins of TaPHS1 were screened by yeast two hybrid system.Three proteins were found to interact with TaPHS1 protein,which are protein kinase(TaPK),voltage dependent anion channel(Ta VDAC)and xyloglucan endotransglycosylase/hydrolase(Ta XTH).(5)The protein of TaPHS1,TaPK and Ta VDAC were located by subcellular localization experiments.The results showed that they were located in nucleus,cytoplasm,cell membrane and mitochondria respectively,but the location of Ta XTH protein was not shown,but the website predicted that the protein might play a role outside the cell.Through the gene function annotation and subcellular localization experiments of the three genes,we know that they may play an important role in signal transduction,energy supply and endosperm weakening in seed dormancy regulation.