Effect Of Brassinolide On The Oil Production Of Brassica Napus L.and The Preliminary Study On Its Mechanism

Brassica napus L.is the most important oil crops in China.Oil production is the main purpose pursued in rapeseed production.Poor light is one of the main reasons for the reduction of rapeseed,and the specific mechanism is still unclear.Brassinolide is an important plant hormone discovered late.At very low doses,it can produce obvious physiological effects,which have important regulatory effects on plant growth and development,but the role and mechanism in B napus L.are still unclear.2,4-Epibrassinolide(2,4-BL)is a highly active brassinolide.In this paper,the effects of 2,4-BL on the growth and development of B napus L.in different growth stages were studied systematically.The effect of 2,4-BL on the oil production of B napus L.was evaluated.Screened important transcription factors involved in brassinosteroid signal and light signal,and cloned and expressed related genes.Through yeast hybridization,Arabidopsis thaliana and rapeseed genetic transformation,the functions of related genes in the regulation of rapeseed oil production by brassinolide were studied.The main results of the study are as follows:1.The growth and development of B napus L.at different growth stages can be effectively regulated by 2,4-BL treatment,2,4-BL promotes oil production mainly by promoting above-ground nutrient growth of rapeseed.At the seedling development stage,1~1000nM/L 2,4-BL promoted seed germination at different levels,and 5~10?M/L 2,4-BL inhibited seed germination.During the seedling development stage,the aerial growth of B napus L.was promoted by 2,4-BL treatment,and the roots were less tolerant to 2,4-BL than the aerial part.During the whole seedling stage,the vegetative growth of B napus L.was promoted by 2,4-BL treatment,and the stem length and leaf area increased significantly.During flowering and pod development,the number of pods in B napus L.and the number of seeds per pod were significantly promoted by 2,4-BL treatment,but continued use of 2,4-BL during the ripening period of pods may lead to prolonged vegetative growth,thereby delaying seed maturation.During the whole growth period,photosynthesis of B napus L.was significantly promoted by 2,4-BL treatment.At the molecular level,the expression of BnaPIF4 and BnaBZR in B napus L.was rapidly induced by 2,4-BL treatment.In general,the effects of 2,4-BL treatment on B napus L.were basically the same,but the effect levels had obvious differences in different rapeseed varieties.2.Two novel PIF4 gene were isolated from Brassica napus L.cv.Xiangyou15,they were identified on chromosomes A03 and C03 and encoding 413 and 414 amino acids,respectively,named as BnaPIF4_A03 and BnaPIF4_C03,their coding sequence(CDS),full–length mRNA and full–length gene were 1242 bp and 1245 bp,1701 bp and 1731 bp,2527 bp and 2665 bp,respectively.BnaPIF4_A03 and BnaPIF4_C03 had seven and eight exons,six and seven introns,respectively.Compared with the sequenced Zhongshuang 11,BnaPIF4_A03 gene had a single base insertion mutation in the first intron,a deletion mutation in the fourth and sixth introns,and a longer 3'–UTR.Other sequences of the two genes did not differ between Xiangyou NO.15 and Zhongshuang11.The BnaPIF4_A03 and BnaPIF4_C03 proteins had a typical plant bHLH domain.The BnaPIF4 protein was highly homologous to the PIF4 protein of Brassica oleracea,Arabidopsis thalian and Eruca sativa.The evolutionary relationship of PIF4 protein was consistent with that of species.PIF4 protein repeats were observed in a large number of plants and the degree of differentiation of PIF4 was lower in lower plants than in higher plants.It indicates that PIF4 protein differentiation is a late evolutionary event and there may be functional redundancy in PIF4 protein.The genes expression patterns of BnaPIF4_A03 and BnaPIF4_C03 in Xiangyou NO.15 were consistent.BnaPIF4 gene was mainly expressed in the green tissue of B napus L.,with higher expression levels in stem epidermis,immature pods and leaves,and lower expression levels in flowers and roots,and the gene expression level of BnaPIF4 gradually decreased in the development process of B napus L.3.Three novel BZR1 coding sequences(CDSs)were isolated from cDNA of Brassica napus L.cv.Xiangyou NO.15 leaves,which were mapped on the chromosomes A07,A06 and A06,and designated as BnaBZR1_A07,BnaBES1_A06F and BnaBES1_A06R,respectively.These three BnaBZR CDSs were 996 bp,993 bp and 996 bp in length and encoded predicted proteins with 331,330 and 331 amino acid residues,respectively.BnaBZR proteins were predicted to be located on the cell nucleus and have a typical plant BZR/BES conserved domain.Multiple sequence alignments and phylogenetic analysis showed that the deduced amino acid sequences of BnaBZR were highly homologous to previously reported BZR/BES of Brassica oleracea,Arabidopsis thalian and Eruca sativa.And the similarity of BZR1 or BES1 among different related species was higher than the similarity between BZR1 and BES1 in the same species or related species,indicating that BZR1 and BES1 differentiation is an early evolutionary event.The expression patterns of BnaBZR1_A07,BnaBES1_A06F and BnaBES1_A06R in Xiangyou NO.15 were similar,whit high expression level at the seedling stage and flowering stage,while slightly lower level at the stage of bolting and podgrain ripening.The expression level of BnaBZR in the aerial parts such as leaves,stems,flowers and pods was higher than that in underground parts.4.The BnaPIF4 and BnaBZR genes were cloned and compared from the two B napus L.varieties XY881 and XY883.It was found that the related gene sequences and structures in XY881 were consistent with the parent material Xiangyou NO.15,but the BnaPIF4 and BnaBZR1 genes in XY883 were different from the parent Xiangyou No.15.There are three different alternative splicing in the first exon of the BnaPIF4 gene in XY883,forming three mRNA containing different 5'-UTRs and having the same CDS.There is a 124 bp long A/T base-rich insertion mutation in the BnaBZR1 gene promoter in XY883.Gene mutations in XY883 affect the corresponding gene expression and regulation,in which the promoter insertion mutation of BnaBZR1 increases the background expression level of BnaBZR1 in XY883,and inhibits the inducing effect of poor light and 2,4-BL on BnaBZR1 gene expression.The three mRNA of BnaPIF4 with different 5'-UTR have significant responses to poor light and 2,4-BL.5.Yeast hybridization experiments showed that BnaPIF4 and BnaBZR/BES proteins interacted with each other to participate in the response of light signals and brassinosteroid signals.BnaPIF4 does not bind to the BnaBZR/BES promoter to regulate its transcription,and BnaBZR/BES binds to the BnaPIF4 gene promoter to affect its transcription.The promoter mutation of the BnaBZR1 gene in XY883 does not affect its interaction pattern and only promotes its expression level.The alternative splicing of the BnaPIF4 gene in XY883 to form three different 5'-UTRs did not affect the transcriptional efficiency of BnaPIF4,but had an effect on the translation efficiency of BnaPIF4 protein.BnaPIF4 is a negative light-regulating factor in both Arabidopsis thaliana and Brassica napus L.,and BnaBZR1 has an inhibitory effect on its negative regulation.The effects of transforming BnaPIF4 and BnaBZR/BES genes were greater in Arabidopsis than in Brassica napus.