| Oilseed rape(rapeseed)belongs to genus Brassica of family Brassicaceae(Cruciferae),and is an important oil crop in China and even in the world.Oilseed rape is the world’s third largest source of vegetable oil after soybeans and palms.In China,oilseed rape is not only the fifth largest economic crop after rice,corn,wheat and soybean,but also the largest oil crop.Rapeseed(Brassica napus,2n=4x=38,AACC)is the main cultivated oilseed Brassica species in China and even in the world.In recent years,many new rapeseed cultivars with high quality and high yield have been obtained through the unremitting efforts of breeders,which have effectively promoted the development of rapeseed industry.However,serious drought stress,high or low temperature,diseases,insect pests,plant lodging and low level of mechanization still restrict the production of oilseed rape.The reason is still caused by low harvest index,less ideal plant morphology and weak adaptability,and the genetic improvement of these aspects is the core pursuit of rapeseed industry.Only the synchronous improvement of these characters can really meet the actual production needs of oilseed rape.However,the effect of a single gene in traditional research generally can only solve one or a few problems,so it is of great significance and prospect to dig out the important key genes which can solve the problems in the above aspects at the same time.MYB transcription factors constitute one of the largest family of transcription factors in plants.It has a large number of genes,a wide variety of types and functions,and is involved in almost all aspects of plant development,growth,metabolism and adaptability.It is involved in the response of plants to biotic and abiotic stresses,regulates cell proliferation and differentiation,histogenesis and organogenesis in the process of plant growth and development,and the types and contents of primary and secondary metabolites in the process of plant metabolism.Therefore,MYB transcription factor has always been a hot spot to study the function of plant transcription factors.Based on comprehensive analysis of a few side/indirect clues from preliminary study of our group and reports of other researchers,we assume that MYB55 in Arabidopsis thaliana or B.napus may be a regulatory factor of plant growth,development and resistance.However,to date there is no systematic cloning and functional study of MYB55 or its orthologous genes in the whole plant research community.Therefore,this study systematically completed the full-length cDNA and gDNA cloning and analysis of nucleic acid and protein molecular characteristics of MYB55 gene family from B.napus and its parent species B.rapa(2n=2x=20,AA)and B.oleracea(2n=2x=18,CC).Their expression patterns,including subcellular localization,organ-tissue specificity,phytohormone responses and adversity inducibility,were detected.Transgenic oilseed rape plants with overexpression of BnMYB55 were obtained.The functional characteristics and possible internal mechanisms of BnMYB55 involved in regulating important traits of B.napus were systematically studied from the aspects of botany,agronomy,physiology,biochemistry,molecular biology,tissue anatomy and histochemistry,transcriptomics and metabolomics of the transgenic plants.1.Cloning and expression characteristics of MYB55 genes from B.napus,B.rapa and B.oleraceaIn this study,members of the MYB55 gene family of B.napus and its parent species,B.rapa and B.oleracea,were cloned.There are two members of MYB55 family in B.napus:BnMYB55-1 and BnMYB55-2.B.rapa and B.oleracea contain only one MYB55 gene in each:BrMYB55 and BoMYB55,respectively.Bioinformatics analysis showed that MYB55 encodes a R2R3-MYB transcription factor of S13 subgroup,which is clustered in neighbor with SAt46,SAt103,S16 and SAt85 subgroups,indicating that these subgroups might have somewhat similarity in structural characteristics or functions.It is speculated that MYB55 might be related to vascular tissue biogenesis and organ lignification.Whether it is B.napus or B.rapa and B.oleracea,all members of the Brassica MYB55 gene family are highly expressed in the stem as revealed by qRT-PCR detection.During the stem developmental process,BnMYB55 family is highly expressed in xylem of mature stem of B.napus.BnMYB55 responded to various stresses and hormone treatments,especially the rapid and significant up-regulation within 0.5 h after inoculation with Sclerotinia sclerotiorum.To sum up,it can be speculated that BnMYB55 is involved in the regulation of growth and development of rapeseed lignified organs and stress resistance.2.Overexpression of BnMYB55 improved the growth and development of oilseed rape organs,and significantly increased the yieldAt the level of agronomic investigation,the growth and development of roots,leaves,stems,branches and grains of transgenic plants were significantly improved,e.g.,the length and number of lateral roots were increased by 54.95%and 54.58%,the leaf area per plant of transgenic plants was increased significantly by 35.42%-62.54%,the diameter of stem of transgenic plants was increased significantly by 20.50%,the number and length of secondary branches of transgenic plants were increased significantly by 366.67%and 173.28%,the numbers of siliques of primary and secondary branches of transgenic plants were increased significantly by 27.20%and 252.66%,the number of seeds per effective silique significantly was increased by 30.18%,and the seed yield was increased by 56.86%,respectively.At the level of tissue anatomy,the numbers of all kinds of cells in the stem crosssection of transgenic plants increased significantly,and the cell wall thickness of xylary fiber and vessel cells increased significantly too.On the physiological level,the net photosynthetic rate and water use efficiency of transgenic rapeseed overexpressing BnMYB55 were significantly higher than WT plants;many hormones in the stem changed with the overexpression of BnMYB55,especially that the gibberellin,brassinolide and strigolactone contents were significantly downregulated in the stem at early bolting stage.On the molecular biology and transcriptomic levels,through qRT-PCR analysis and RNA-seq analysis,it was found that many important genes related to cell cycle,cell wall biogenesis and developmental processes were all up-regulated.To sum up,no matter at the agronomic level,physiological level,tissue anatomy level or molecular biology level,overexpression of BnMYB55 promoted the growth and development of oilseed rape(especially the histogenesis and growth of vascular organs),improved the plant morphology,and then increased the yield of oilseed rape.3.Overexpression of BnMYB55 significantly improved the lodging resistance of oilseed rapeAt the level of agronomic investigation,the root system of transgenic rapeseed was more developed,the stem was stronger,and the ratio of root to shoot increased.At the physiological level,the breaking-resistance strength of transgenic plants was greatly improved,especially in the lower part of the stem of transgenic plants it was significantly up-regulated by 39.52%.On the level of tissue anatomy and histochemistry,cross-section area of the stem increased greatly,the area of pith and xylem of transgenic oilseed rape increased correspondingly,the thickness of lignified tissue increased by 27.54%,and the cell wall thickness of xylary fiber and vessel cells increased by 33.39%and 26.82%respectively.At the biochemical level,the content of cellulose,lignin and pectin in the cell wall of the stem of transgenic plants was significantly increased by 10.03%,11.47%and 25.69%,respectively.At the level of molecular biology,important genes related to cellulose,lignin and pectin biosynthesis were up-regulated in transgenic plants.At the transcriptomic level,for RNA-Seq data,GO analysis found that in Cellular Component the DEGs of each organ were enriched on Plant-Type Cell Wall.The results of KEGG Pathway Enrichment analysis showed that the DEGs of each organ were enriched in the phenylpropanoid biosynthesis pathway.To sum up the results from above layers,overexpression of BnMYB55 significantly improved the lodging resistance of oilseed rape.4.Overexpression of BnMYB55 significantly improved the resistance of oilseed rape to S.sclerotiorumOn the physiological level,through the inoculation of S.sclerotiorum,the results showed that the area of leaf lesions and the length of stem lesions of transgenic plants decreased significantly by 23.37%and 70.96%,respectively.At the level of tissue anatomy and histochemistry,the xylem area of transgenic plants increased,the xylem was thickened significantly by 27.54%,and the cell wall thickness of xylary fiber and vessel was thickened significantly by 33.39%and 26.82%respectively.At the biochemical level,the content of cellulose,lignin and pectin in the stem of transgenic plants was significantly increased by 10.03%,11.47%and 25.69%respectively.At the level of molecular biology,through qRT-PCR analysis it was found that major genes related to cellulose,lignin and pectin biosynthesis were up-regulated in transgenic plants.In addition,the genes related to the induction by S.sclerotiorum,such as WRKY18,NINJA,WRKY28,WRKY75,WRKY33 and WRKY22,were changed significantly towards to increasing resistance.At the transcriptomic level,RNA-Seq data and GO analysis indicated that in Biological Process the DEGs of each organ were enriched in response to various stresses,in Cellular Component the DEGs in roots,leaves,flowers and seeds were all enriched into plant-type cell wall,and in Molecular Function the enrichment of various organs tended to participate in the regulation of plant secondary metabolism,resistance to fungal diseases and stresses.The results of KEGG Pathway Enrichment analysis showed that the DEGs of each organ were enriched in the interaction pathway between plant and pathogen.The secondary metabolites in stems,leaves and seeds of transgenic oilseed rape were determined.The results showed that overexpression of BnMYB55 caused quantitative remodeling of phenylpropanoid biosynthesis flux and significant changes in glucosinolate biosynthesis pathway.In summary,overexpression of BnMYB55 significantly improved the resistance of oilseed rape to S.sclerotiorum.5.Overexpression of BnMYB55 reshaped and enhanced the plant growth-defense trade-offsPlant defense needs secondary metabolites,and the production of secondary metabolites needs matter and energy,and the plant itself is limited by resources and assimilation products.If there are more resources allocated for resistance,there should be relatively less resources for plant growth and development and seed production,a tradeoff phenomenon in plant growth-defense relationship.But we know very little about the regulatory mechanism between growth and defense.It is a challenge for crop breeding to improve disease resistance and yield potential at the same time.In this paper,overexpression of BnMYB55 can not only improve the growth and development and plant morphology,increase yield,but also increase the ability of resistance to lodging and S.sclerotiorum in oilseed rape simultaneously.This is not only related to growth-defense trade-off in utilization of limited assimilates,but also related to source broadening,i.e.higher strength of assimilates accumulation,because both net photosynthesis rate and water use efficiency also increased significantly in BnMYB55-overexpressing plants.BnMYB55 can be used as a new key regulator of remodeling and enhancing growthdefense trade-offs,and a new strategy to simultaneously obtain high disease resistance,high lodging resistance,ideal morphology,high biomass and high grain yield can be explored by studying the function of BnMYB55 genes.This study systematically revealed the functional characteristics and primary internal mechanism of BnMYB55 gene family involved in a variety of metabolisms and important traits in oilseed rape,which laid a good foundation for further studying its deep mechanisms,and showed great potential in crop molecular breeding. |
