The Relationship Between Drought-tolerance/Fracture-resistance And Lignin And Its Corresponding Molecular Mechanism In Brassica Napus L.

Brassica napus L.?rapeseed?is the largest oil crop in China.In recent years,with the improvement of varieties and the supporting agronomic technology,the production area and yield of rapeseed have gained more space for expansion.But at present,the main limiting factors that block the potential of rapeseed production in China include weak resistance and low mechanization.Seasonal drought,which is often encountered in the main producing areas of the Yangtze river,is one of the main factors contributing to the yeild loss.In addition,with the continuous development of modern breeding techniques and improved varieties,rapeseed production is increasing.However,the increase in yield also caused an augment in the weight of parts of the above ground,which in turn increased the support pressure on the stems of rapeseed,and could easily cause lodging,ulteriorly induced the reduction of yield and difficulties of mechanical harvesting.Therefore,studying the mechanism of drought resistance and lodging resistance in rapeseed,identifying genes related to resistance and clarifying the regulatory network can provide important theoretical basis for rapeseed resistance breeding.This study carried out from the two aspects,namely drought resistance and lodging resistance of rapeseed.Firstly,by investigation of the phenotypic traits of rape under drought stress,we intended to understand the effects of drought stress on vegetative growth and reproductive growth,and to analyze the relationship between traits in response to drought stress andfurther screening rape drought-resistant varieties;Secondly,we analyzed the drought resistance mechanism of rape from the transcriptional level,and identify important genes for drought resistance;Moreover,using soft-stem mutants to analyze the lodging resistance mechanism of rapeseed transcription level.The specific findings are as follows:1.Effects of drought stress on agronomic traits of Brassica napusIn this study,121 genetically diverse materials were selected from 500 germplasm resources and grown in rain-protection shed withdrought-treatment along with controls with normal irritationThe growth period,biological yield,relative chlorophyll content,pod-related traits and lodging-related traits of rape were investigated at the early flowering stage or mature stage and were statistical analysed,the results show that:drought stress can advance flowering time and extend the flowering phase to a certain extent.Meanwhile,drought stress has negative effects on most agronomic traits.Plant growth is retarded under drought stress,and this impact is more significant among rapeseed varieties which possess vigorous vegetative growth and greater biological yeild.Under drought stress,the fracture strength and lignin content of rape stem increased significantly.The correlation analysis showed that plant height deviation was significantly positively correlated with effective length of main inflorescence and effective branch height deviation at mature stage.The negative correlation between effective length of main inflorescence deviation and primary branch number deviation was extremely significant.The correlation between the deviation of silique number of main inflorescence and the silique length was negatively significant.The dry weight deviation of the plants was positively correlated with the fracture resistance deviation and significantly correlated with the diameter deviation of the stem.There was no significant correlation between the deviations of stem diameter and the fracture resistance.There were no significant correlations between the deviation of lignin content and dry weight,stem diameter and fracture resistance.The stem fracture strength and lignin content were significantly correlated under drought stress and not related under natural conditions,suggesting that lignin is associated with rape lodging resistance under drought stress.In all,it is indicated that moderate control of water is beneficial to improving the lodging resistance of rapeseed.2.Analysis of differential gene expression induced by drought stress in B.napusIn this study,by investigated the agronomic traits of rapeseed under drought stress,we selected five materials with high lignin content and five low lignin content materials respectively with higher drought tolerance,and analyzed similarities and differences of the gene expression of high and low lignin materials under drought stress.The results are as follows:after comparing with the FPKM of high CK and low CK,we looked for the differentially expressed genes?DEGs?with a difference of at least 2 times?|log₂?W2/W1?|?1,FDR<0.01?,and there were 533 and 1,516 DEGs associated with drought stress in high/high CK?High?and low/low CK?Low?respectively.328 genes were up-regulated and 205 were down-regulated in High,and 262 genes were up-regulated along with 1254 genes were down-regulated in Low.It illuminated that plants with high lignin content were more resistant to drought stress accompanied by less drought-induced DEGs,but more DEGs were activated in low lignin content materials to withstand the water-deficient threat by up/down regulation.DEGs were analyzed by GO enrichment.The results showed that upregulation of DEGs was mainly located in the plasma membrane and vacuole and involved in the process of stress response.Down-regulation of DEGs was mainly located in the cell wall and extracellular regions and was mainly involved in processes of cell development,reproduction,cellular components derivation,localization,and transfer.The same up-regulated genes in High and Low are mainly involved in the rhythm process,transport process,and biological regulation,among which,circadian rhythm?GO:0007623?,rhythmic process?GO:0048511?,long-day photoperiodism?GO:0047571,GO:0048574?,and regulation of auxin metabolic process?GO:0090354?,regulation of auxin biosynthetic process?GO:0010600?and regulation of hormone metabolic process?GO:0032350?were only up-regulated in High,and specific upregulated genes in Low were mainly involved in developmental processes,secondary metabolic processes,cellular metabolic processes,and small-molecule metabolic processes,suggesting that the drought stress response was related to circadian rhythms and transport,while the degree of lignin accumulation affected circadian rhythms andhormone metabolism.There was a large difference in the number of DEGs between High and Low.Up-regulated GOs in Low were up to 3 times of that GOs in High and downregulated DEGs reached up to 10 times.KEGG enrichment analysis showed that the same DEGs in High and Low were up-regulated in circadian rhythm-plant?ath04712?,which was consistent with the GO analysis results.In addition,the simultaneous downregulation pathways in High and Low included pentose and glucuronate interconversion?ath00040?,ascorbate and aldarate metabolism?ath00053?,fatty acid degradation?ath00071?,phenylalanine metabolism?ath00360?,tryptophan metabolism?ath00380?,phenylpropanoid biosynthesis?ath00940?.DEGs were mainly enriched in secondary metabolic pathways.which were up to 10 times higher than other pathways.The high and low specific metabolic pathways were mainly concentrated on amino acid,carbohydrate metabolism and lipid metabolic pathways.High and Low had 43 and 55transcription factors,respectively.MYB/MYB-related,MIKC and NAC were the major three types of transcription factor family induced by drought stress.3.Cloning and evolutionary relationship of the caffeic acid-O-methyltransferase genes in B.napusThrough high-throughput transcriptome sequencing,it was found that there were differences in the expression of several members of the COMT gene family in the lignin synthesis pathway under drought stress.In this study,the identification and phylogenetic analysis of the COMT genes of four cruciferous plants?Arabidopsis,Brassica rapa,Brassica oleracea,and Brassica napus?were performed to identify important family members of lignin biosynthesis and analyze the tissue and spatio-temporal expression specificity of these members.The results are as follows:Based on the AtOMT1 gene and 13 COMT-like genes annotated in the TAIR database,the CDS sequences were aligned in the Brassica napus genome database and Brassica genome database to identify candidate B.napus COMT genes.There were 42 B.napus CDS sequences,22 Brassica rapa,and 20 Brassica oleracea were comfirmed as COMT genes.The 12 BnCOMT1 genes homologous to Arabidopsis OMT1?AT5G54160?were identified and 5 of them were cloned for ORF sequences and their corresponding genome sequences to verify the accuracy of the database information.The DNA of the full-length genes of the BnCOMT1 family included 4 exons and 3 introns,among which,intron 1 had the greatest variation,and introns II and III are relatively conserved.BnCOMT1 were acidic proteins.There are 21-37 potential phosphorylation sites.Except BnCOMT1-1,each BnCOMT1 contained a Winged helix-turn-helix DNA-binding domain?IPR011991?,a methyltransferase dimerisation domain?IPR012967?and a S-adenosyl-L-methionine-dependent methyltransferase domain?IPR029063?.Phylogenetic analysis showed that cruciferous COMTs were divergent during evolution and clustered into five major groups.Based on the functional annotation of the existing AtOMT genes,it was speculated that the COMTs in groups C,D,and E were mainly involved in lignin biosynthesis.Group A was mainly involved in the metabolic pathway of glucosinolates.The evolution order of COMT methyltransferase₂ domains and full-length genes were different in various species,indicating that there is an inequality between the evolution of domain region and the full-length genes.Group E and group D were considered to be flavonoids-O-methyltransferase and O-diphenol-O-methyltransferase proteins which were involved in the biosynthesis of lignin monomers and phenylpropane,respectively.In addition,the protein identity and similarity among the main members of BnCOMT1s were roughly the same as their evolutionary relationships,and their protein identities were relatively high among genes with similar evolutionary relationships.The intron I of the BnCOMT1 gene family is extremely conserved and remains highly consistent with its parental species in evolution.BnCOMT1s were expressed in all organs examined,among which BnCOMT1-4,BnCOMT1-8,BnCOMT1-5 and BnCOMT1-10had the highest expression levels in stems,and their expression levels reached 3 times of their levels in other organs.The expression levels of BnCOMT1-4,BnCOMT1-8,BnCOMT1-5,and BnCOMT1-10 in the root were only slightly higher than those in other organs,indicating that the expression of BnCOMT1-4,BnCOMT1-8,BnCOMT1-5,and BnCOMT1-10 was not constitutive expressed and was related to the lignification of the plant.The expression of BnCOMT1-2 in the root was also relatively high and was only lower than that of the 15D seed.The genetically similar members of BnCOMT1-4,BnCOMT1-8,BnCOMT1-5,BnCOMT1-10,and BnCOMT1-2 showed similar tissue-specific expression.BnCOMT1-3,BnCOMT1-9,and BnCOMT1-6 had higher expression levels in leaves,while BnCOMT1-11 and BnCOMT1-12 had higher expression levels in floral organs and 45D seeds,respectively.BnCOMT1-3,BnCOMT1-9,and BnCOMT1-6 were barely expressed in 30D seeds,but continued to be expressed in 45D seeds.Under natural conditions,the transcript abundance of stem-specific expression BnCOMT1 genes?BnCOMT1-4,BnCOMT1-5,BnCOMT1-8,and BnCOMT1-10?was positively proportional to the total lignin accumulation,that is,the gene expression level of high lignin materials was also relatively high,and BnCOMT1s were expressed in all stages of development under natural conditions.The peak of expression occurred at the early flowering stage,while under drought conditions,the peak was delayed to the final flowering stage.4.Analysis of differentially expressed genes in soft stem materials of Brassica napusIn this study,rapeseed varieties with weak stem mechanical strength and curving stem during bud and early flowering stages were selected by years of field observation as?soft-stem varieties?,and high-throughput RNA-Seq technology was used to perform transcriptome sequencing analysis of soft-stem varieties and normal varieties.Thus,stem-strength-related genes and their associated biological processes and molecular functions were analyzed at the transcriptional level.The results showed that RNA-seq high-throughput sequencing yielded FPKM of normal stalk?N?and soft-stem?S?expression.By searching for FPKM expression values that differed by at least 2-fold?|log₂?S/N?|?1,FDR<0.01?,there were 1251 differentially expressed genes related to soft stems,of which 590 genes were up-regulated and 661 genes were down-regulated.The difference in the expression levels of 158 genes was more than 16times,of which 87 were up-regulated and 71 were down-regulated.There were 10 genes whose expression varied 64 times or more.Among them,4 genes were up-regulated and6 genes were down-regulated.The GO function annotation of DEGs showed that most of the genes in cellular components were up-regulated and distributed in each cell component,and their major positions were in the extracellular region?GO:0005576?and chloroplast?GO:0044434?,whereas the down-regulated genes were located only in the extracellular region?GO:0044421,GO:0005615?.In terms of molecular function,differential genes were mainly related to catalytic activity?GO:0003824?.In terms of biological processes,stem intensity-related genes were mainly involved in stress response?GO:0050896?,cellular processes?GO:0009987?,and metabolic processes?GO:0008152?,In addition,endogenous stimuli response?GO:0009719?,signal transduction?GO:0023052?,immuneresponse?GO:0006955?,localization?GO:0051234?,stress response regulation?GO:The genes of 0048583?and immune system process regulation?GO:0002682?were only down-regulated.Through the statistical analysis of GO enrichment of DEGs,the DEGs related to stress response were the most active,with 185 up-regulated DEGs and 213 down-regulated,and their mainly stress responses included response to stress?GO:0006950?,response to abiotic stimulus?GO:0009628?,and response to chemical stimulus?GO:0042221?.The up-regulated KEGG metabolic pathways were 2-oxo carboxylic acid metabolism?ath01210?,phenylalanine metabolism?ath00360?,glucosinolate biosynthesis?ath00966?,valine/leucine/isoleucine biosynthesis?ath00290?and sulfur metabolism?ath00920?.The down-regulated pathways mainly included plant hormone signal transduction?ath04075?,plant-pathogen interaction?ath04626?,phenylpropanoid biosynthesis?ath00940?,?-linolenic acid metabolism?ath00592?and Linoleic acid metabolism?ath00591?.The results of this study provide a better foundation for the further excavation of stem fracture-resistance genes in rapeseed.