| Plant growth and development are affected by a variety of environmental factors.With the global climate change,frequent and serious drought stress on food security has attracted more and more attention.Haynaldia villosa（VV,2n=14）,as a tertiary gene bank for wheat genetic improvement,shows excellent tolerance to drought stress.Analysis of the molecular mechanism of drought resistance in Haynaldia villosa will help to explore its elite drought resistance gene resources which could be used for the improvement of wheat drought tolerance in genetic breeding.RNA-seq is a high throughput sequencing technology that detects the presence and quantity of RNA in tissues at a specific time.Using this technology combined with the analysis of differentially expressed genes can effectively investigate the molecular mechanisms of phenotypic changes.In this study,RNA-seq technology was used to discover the comprehensive mechanism involved in the drought tolerance in Haynaldia villosa accession 91C43 under simulated drought stress using PEG6000.The transcriptome sequencing（RNA-seq）was performed on the eight samples,including leaves and roots at 0h,6h,24h,48h under stress treatment respectively,and three biological replicates were performed.Mitogen-activated protein kinase（MAPK）cascade signaling pathway in plants is a very important signal transduction pathways involved in a wide range of biological processes,such as growth,development and stress responses.Haynaldia villosa has confers resistance to various diseases and tolerance to different abiotic stresses.So,it is of great significance to study the mechanism of MAPK cascade signaling pathway in Haynaldia villosa in stress resistance.Previuosly,our laboratory found several MAPK genes resoponsive to drought stress and cloned them in Haynaldia villosa.However,the mitogenactivated protein kinase（MAPKK or MKK）which located upstream of the signal cascade have not been cloned and the construction of MAPKK-MAPK signal network has not been carried out.This study used genomic information and transcript information to carry out identification and bioinformatics analysis of MAPKK family members for multiple species.Then,MAPKK family members cloning,gene and protein structure analysis,gene evolution study,MAPKK-MAPK cascade interaction network construnction was performed in Haynaldia villosa.1.RNA-seq data acquisition and quality assessment:A total of 24 samples from 2 tissues,4 time periods,and 3 replicates were subj ected to RNA-seq sequencing under drought stress.A total of 155.45 Gb of raw data was obtained.Clean Reads of each sample reached to 93.69%;the sequencing quality of Clean Reads reached to Q7.25%and Q30 reached to 89.40%;the mapping rate of Clean Reads reached to 91.57%.The data quality meet the requirements of subsequent quantitative comparison analysis;90263 unigene were was assembled,with the average length,N50 and GC content to be 1344 bp,1903 bp and 49.47%.Totally,2080 transcription factors were identified.2.Analysis of the molecular mechanism of drought resistance of Haynaldia villosa based on differentially expressed genes:There is no obvious correlation between the root and leaf gene expression of Haynaldia villosa.After 6 hours of drought stress,the leaves quickly responded to drought stress signals,and the expression levels of genes related to stomata closure,mismatch repair,and trehalose synthesis pathway were significantly increased.The genes related to ABA signaling pathway and H2O2 response pathway were activated rapidly in roots,which reduced the damage of ROS.The expression of genes related to amino acid metabolism,ethylene synthesis,sugar and raffinose metabolism,protein folding and other pathways in leaves increased significantly at 24 hour of drought stress,and in root ABA signaling pathway was continuously activated,JA signaling pathway and glutathione metabolism were newly activated.At 48 hours of drought stress,the leaves activate arginine and proline metabolism to increase the intracellular water potential,activate the synthesis of horns and waxes to form a water-retaining layer outside the leaves,and use glutathione metabolism pathways to reduce intracellular activity Oxygen level.In root,the arginine and proline metabolism pathways were also significantly activated,the synthesis pathways of small molecule osmotic adjustment substances such as fructose and mannose were activated,and the genes related to the synthesis of non-enzymatic ROS scavenging substances,such as ascorbic acid and vitamin B6,were activated.Linolenic acid and other lipid metabolism pathways were activated to enhance the fluidity of cell membranes and maintain the stability of membrane functions.3.Bioinformatics analysis of MAPKK family members:（1）The number of MAPKK family members:There is a correlation between the number of MAPKK genes and the ploidy of species,for example,the number of MAPKK in diploid Arabidopsis thaliana,rice,Brachypodium distachyon other diploid species were between 10-13.The number of triticeae crops MAPKK varied correspondingly with the ploidy,for example,the number of MAPKK in 10,26 and 40 in Triticum urartu,Triticum dicoccoides and Triticum aestivum,respectively.（2）Classification of MAPKK family members:The MAPKK family can be divided into four groups as A,B,C and D.The MAPKK family in Gramineae is relatively conservative.However,there was significant differentiation between the dicot Arabidopsis and monocot Gramineae species.（3）Structure of MAPKK family members:The structure of MAPKK family genes differs greatly between groups.Members of group A and group B have multiple exons and the intron length varies greatly between different species.There are no introns in groups C and D except for individual genes.（4）Chromosome distribution of MAPKK family members:Physical location analysis of wheat MAPKK family members showed good collinearity in the genomes of Tritcum aestivum,Triticum dicoccoides,Aegilops tauschii and barley.Fitting the position of the MAPKK family members on the chromosome on a scale of relative length 1,the distribution of members of each group on the chromosome were overlapped well.The distribution of MAPKK groups on some homoeologous groups has a certain preference.For example,group A is distributed in the 4th,6th and 7th homoeologous groups,while group B is distributed in the 5th homoeologous group,group C is distributed in the 6th and 7th homoeologous groups and group D is distributed in the 1st and 4th homoeologous groups.（5）Expression patterns of MAPKK family members:Using the wheat public expression database,the expression patterns of wheat MAPKK family members were analyzed.It was found that there were significant differences in the expression levels between different groups,most of D members were not expressed,while the A members shows the highert expression level followed by B and C members.It was worty noted that some MAPKK genes can be activated by various stresses.4.Cloning of MAPKK and construction of MAPKK-MAPK signal transmission network in Haynaldia villosa:Biological analysis of the transcriptome data of Haynaldia villosa identified 9 MAPKK genes,which could be divided into 4 groups as A to D.Amino acids in length was between 348 to 930 and an isoelectric point was between 5.33 to 9.4.MAPKK family contains 11 conserved domains and conserved S/TxxxxxS/T motifs necessary for activation of MAPKK.According to the gene family analysis,primers were designed for amplification of MAPKK gene and one gene was newly cloned from Haynaldia villosa.Using yeast two-hybrid technology,interaction between 4 MAPKK and 14 MAPK were studied,then it was found that MAPKK3-V interacted with MAPK2-V and MAPK4-V,and MAPKK6-V interacted with MAPK1-V and MAPK3-V.5.Identification of resistance to PEG6000 in mapkll-v transgenic plants:Positive transgenic plants were identified in the T2 generations of MAPK11-V transformed plants,and eight positive lines derived from four To indiveduals were obtained.The seedling drought resistance of trangenic plants in T2 generation and T3 generation of two independent lines were evaluated under the stimutaed drought stress by treatment with PEG6000（20%）.The results show that the wilting degree of the first/second/third leaves was lower than that in Yangmai158,whilt the length of the fourth/fifth leaves were longer than that of Yangmai 158.As to the physiological parameters,net photosynthetic rate,stomatal conductance and the maximum photochemical efficiency of photosystem II of the transgenic lines were higher than that in Yangmai158.It is speculated that MAPK11-V can paritially improve to some extent drought tolerance of wheat in seedling stage under PEG6000（20%）stress. |
