Regulation Of Plant Growth And Development By Key Genes Of Brassinosteroids Anabolism

Brassinosteroids(BR),also known as brassinosteroids,is a plant hormone identified in recent decades.Although its content is relatively low in plants,it plays an important role in regulating plant development and morph formation.As a phytosterol endogenous hormone,BR plays an important regulatory role in plant growth and development,vascular system differentiation,cell division,etc.,because its physiological and biochemical activity is far more than the existing five hormones(auxin,gibberellin,cytokinin,ethylene,abscisic acid),it has been praised as the sixth hormone in the world.Moreover,a large number of studies have demonstrated that BR also plays an important role in plant response to biotic and abiotic stresses.The synthesis and signal transduction pathway of BR is an extremely complex process.There are early and late pathways in the synthesis of BR,which are C-22 oxidation pathway,C-6 oxidation pathway and the combination pathway between them.In the whole C-6 oxidation pathway,CYP85A,as an important gene,participates in the biosynthesis process of BR.It also plays an important role in the activity of BR.At present,it has been proved that exogenous brassinosteroids can significantly improve the tolerance of crops to various environmental stresses,such as heat stress,cold stress,drought,etc.,but there is a lack of research on endogenous BR tolerance to stress.Therefore,this study took tomato as experimental material and created transgenic tomato with overexpression of BR synthetic gene CYP85A to explore the effect of endogenous increase of BR on plant response to stress.Firstly,we amplified tomato SlCYP85A gene using Money Maker wild-type tomato cDNA as template.Subsequently,SlCYP85A overexpression lines were obtained by Agrobacterium-mediated transformation,and two transgenic lines L4 and L18 were selected as experimental materials for further study of SlCYP85A function according to different up-regulated expression levels.After planting,the selected L4 and L18 strains and wild-type tomato(WT)were treated with salt and drought respectively,and the biomass,ion content,physiological indexes related to active oxygen species and expression levels of active oxygen-related genes of tomato strains of different genotypes were analyzed.The specific research results are as follows:1.SlCYP85A overexpression vector was constructed to obtain SlCPY85A overexpression tomato lines by means of tomato genetic transformation system,and two tomato transgenic lines L4 and L18 were screened according to the differences in expression levels.2.A large number of transgenic plants will be obtained through callus culture,subgeneration and seedling cultivation.3.SlCPY85A overexpressed transgenic plants and wild-type plants with the same growth cycle were used as experimental materials to carry out natural dehydration and drought treatment and 200 m M sodium chloride simulated salt treatment.Ion content,ROS related indexes and expression levels of ROS related genes in leaves of plants treated for 8 days were analyzed.4.The results showed that,compared with wild-type plants,SlCPY85A overexpressed transgenic plants L4 and L18 showed enhanced stress resistance,indicating that tomato SlCPY85A had the biological function of enhancing plant tolerance to stress.BR synthesis requires complex and ordered signal transduction pathways to regulate plant growth and development.In BR signal transduction,the BR receptor on the surface of plant cells can feel the external stimulation,and then affect the physiological and biochemical processes of the cells through the internal signal transduction mechanism.At the end of BR pathway exists a very important transcription factor,BES1,which plays a crucial role in BR signal transduction.At present,the signaling pathway of BR in herbaceous plants is basically clear.However,the BR signaling pathway in woody plants is not perfect enough.Therefore,this study will take poplar,a woody plant model species,as the experimental material to explore the role of PtBES1 genes in the growth and development of poplar.In this study,14 poplar PtBES1 genes were identified and screened from the whole genome library by BLAST and HMM.Bioinformatics analysis was conducted on the gene family members,and gene structure,conserved motif and promoter elements were analyzed.Based on the research of Arabidopsis Thaliana on the negative regulation of BEH3 on vascular tissue development in BES1 gene family,the expression of BEH3 genes in poplar tissues was analyzed in this study.The expression of PtBES1 members in poplar stems and leaves treated with exogenous BR was analyzed,and it was found that PtBEH3b and PtBEH3c responded to BR treatment.In order to further explore the function of PtBEH3b and PtBEH3c in poplar growth and development,this study takes PtBEH3b and PtBEH3c as the target to explore the function of BEH3 genes in poplar growth and development.Specific research results are as follows:1.A total of 14 PtBES1 genes were identified in the whole genome database of poplar,which were divided into four subfamilies according to their similarity in protein structure and phylogenetic relationship.Meanwhile,conservative motif and gene structure analysis showed that the information of gene structure of each subfamily gene was relatively conserved.2.The expression changes of PtBES1 gene were analyzed after exogenous BR treatment,and it was found that the expression levels of most PtBES1 genes changed,among which the expression levels of PtBEH3b and PtBEH3c had the most drastic changes.Therefore,PtBEH3b and PtBEH3c were selected for further study in this study.3.The subcellular localization of PtBEH3b and PtBEH3c was analyzed by tobacco transient transformation experiment,and it was found that both PtBEH3b and PtBEH3c were located in the nucleus and confirmed the identity of their transcription factors.4.Create poplar plants with overexpression of PtBEH3b and PtBEH3c;The gene editing vector was constructed.5.Through the analysis of yeast two-hybrid screen library experiment,we speculated that PtBEH3b and PtBEH3c might co-regulate wood formation process by interacting with key genes in wood development.