| Wheat is an important staple food crop in the world,and its production is related to food security.It is of great significance to explore the key regulatory genes and their mechanisms in the process of wheat growth and development and stress response for wheat production and food security.micro RNAs(miRNAs)are an abundant class of small,single-stranded,noncoding RNAs(21-24 nt in length)found in eukaryotes;many miRNAs are effective posttranscriptional regulators that play important roles in regulating biological processes such as plant growth and development,and responses to biotic and abiotic stresses.Our research team constructed a small RNA library of Xiaoyan 6 wheat in the early stage,and identified some species-specific miRNAs through deep sequencing analysis,including tae-miR5048,which is unique to wheat.However,the gene locus of miR5048 in the wheat genome,the target genes regulated by miR5048,and its role in wheat growth and development and stress response are still unclear.In this study,the target genes regulated by tae-miR5048 were identified and verified by bioinformatics prediction,degradome analysis,RLM-5’RACE,and transient coexpression analysis of tobacco leaves;Transgenic overexpression and CRISPR/Cas9 technologies were used to create tae-miR5048 and its target gene overexpression wheat lines and their loss-of-function mutants,and to investigate the phenotypic changes of these wheat materials under growth,development and stress,to reveal the biological functions of taemiR5048 and its target genes;At the same time,the mechanism of tae-miR5048 and its target genes in wheat growth,development and stress response was analyzed by yeast two-hybrid screening of wheat c DNA library and multi-protein interaction technology.The main conclusions of this study are as follows:1.There are three miR5048 gene loci(TaMIR5048-7A,-7B and-7D)in the wheat genome,which produce the same mature sequence;tae-miR5048 targets and regulate the expression of TaMAPK1 and TaNAK1 in wheat.TaNAK1.1,TaNAK1.2 and TaNAK1.3 all have kinase domain and NB-ARC domain,but the location of the two domains and the length of the kinase domain are different among the three;only TaNAK1.1 and TaNAK1.2 alternative splicing variants are detected in wheat.2.miR5048,TaMAPK1 and TaNAK1 are expressed in all tissues and organs of wheat.The expression level of tae-miR5048 was higher in young spike and grain at the early development stage,and the expression level of TaMAPK1 was higher in leaves.The expression of TaMAPK1 and TaNAK1 was significantly upregulated under P.striiformis infection,while the abundance of tae-miR5048 was decreased under P.striiformis infection,and the expression of TaMAPK1 and TaNAK1 was significantly upregulated under abiotic stresses(drought,high salt,low temperature,Me JA and ABA).The temporal and spatial expression patterns of TaNAK1.1 and TaNAK1.2 are different from those under biotic and abiotic stresses.TaMAPK1 was localized in the cytoplasm and nucleus;TaNAK1.1 was localized in the cytoplasm,and TaNAK1.2 was localized in both the cytoplasm and nucleus.3.Overexpression of miR5048 significantly inhibited the expression levels of TaMAPK1 and TaNAK1,resulting in early heading and flowering,increased plant height and grain size,but no significant changes in grain weight and yield per plant;TaMAPK1 negatively regulate that yield-related traits and yield potential of wheat,TaMAPK1 overexpression result in a reduction in plant height and spike length by 4.0%-4.7% and 13%-19%,respectively,a reduction in grain length and width by 3.6%-4.6% and 0.8%-2.5%,respectively,and a reduction in grain weight by 5.0%-8.4%,As a result,the yield per plant decreased by 16.8%-26.3%;The heading and flowering time of the TaMAPK1 mutants mapk1-aa and mapk1-bb were advanced about 7 days,and the plant height,flag leaf area and spike length were increased by 9.4%-12.4%,56.8%-104.3% and 26.4%-34.0%,respectively.The grain length and weight were increased by 1.9%-5.4% and 8.4%-15.1%,respectively,resulting in an increase of about10% in yield per plant.4.Under the infection of CYR31,the resistance of miR5048-overexpressing wheat was decreased,and the resistance of TaMAPK1 or TaNAK1.2 overexpressing wheat was increased compared with that of wild-type wheat Fielder.Transcriptome analysis showed that under the infection of wheat stripe rust pathogen,TaMAPK1 overexpress signal transduction related protein(including plant sulfur factor receptor 2,serine-threonine protein kinase,acid phosphatase and calmodulin 5/6/7/8,etc.),transcription factors(including TaWRKY35,TaWRKY70 and b ZIP transcription factor TaTGA2.1,etc.),and wheat pathogen resistancerelated genes(including NBS-LRR genes RPM1,TaNPR2,TaNPR3 and TaPR1)were significantly up-regulated.5.Under abiotic stresses(drought and cold),drought tolerance and cold tolerance of TaMAPK1-OE or TaNAK1.2-OE wheat were significantly enhanced compared with Fielder.Transcriptome analysis showed that 708 DEGs were identified between TaMAPK1-OE wheat and wild-type Fielder under drought stress,of which 388 were up-regulated and 320 were down-regulated;Go enrichment and KEGG analysis showed that these DEGs were mainly involved in plant hormone signaling,MAPK signaling,phosphatidylinositol signaling,ABC transporters,transcription factors,abiotic response and other biological processes and metabolic processes.6.By yeast two-hybrid screening of wheat c DNA library and several protein-protein interaction techniques(Bi FC,Y2 H,LCI and Pull-Down,etc.),TaMAPK1 was identified and proved to be interact with TaMAPK1 kinase 2(TaMAPKK2),protein phosphatase 2C(TaPP2C1),transcription factors TaTGA2.1 and TaICE2;TaMAPKK2 interacts with TaMAPKK18.7.Based on the above results,a working model of wheat-specific miR5048-TaMAPK1/TaNAK1.2 module was proposed to coordinate the balance between wheat growth and development and stress resistance;The miR5048-TaMAPK1/TaNAK1.2 module carries out signal transmission of growth and development or adversity stress through a TaMAPKKK18-TaMAPKK2-TaMAPK1 cascade pathway,And coordinate that balance between the growth and development of wheat and the stress resistance through the interaction of TaMAPK1,TaMKP1,TaPP2C1 and transcription factors TaTGA2.1 and TaICE2,thereby promoting the expression of growth and development or stress resistance related genes.In conclusion,three homoeologs of miR5048 are located on homologous group VII chromosomes,which target the expression of TaMAPK1 and TaNAK1.miR5048 positively regulates wheat heading,flowering and plant height,and negatively regulates wheat resistance to stripe rust;TaMAPK1 and TaNAK1.2 negatively regulate wheat plant height,spike length,grain size,grain weight and yield per plant,and positively regulate wheat resistance to biotic stress(stripe rust)and abiotic stress(drought and cold);The cellular mechanism of miR5048-TaMAPK1/TaNAK1.2 module regulating wheat grain size and the molecular mechanism of miR5048-TaMAPK1/TaNAK1.2 module involved in wheat stripe rust resistance,drought resistance and cold resistance were preliminarily revealed;A working model of miR5048-TaMAPK1/TaNAK1.2 module was proposed to coordinate wheat growth and development and stress resistance. |
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