Cloning And Functional Analysis Of Transcription Factors BZR1 And BES1 In Broccoli

Brassica oleracea was one of the most imported vegetables in developed countries and contained the most active natural active substance?sulforaphane,which had so far been found in vegetables.The synthetic precursor of sulforaphane wasglucosinolate,a secondary metabolite of nitrogen and sulfur,which was widely found in cruciferous plants and was important for plant response to biological or abiotic stresses,and its part of the degradation had a strong health care function on human.Therefore,its synthesis and regulation of molecular biology in recent years had gradually become a hot research.Brassinosteroids(Brsinosteroids,BRs),as a plant-specific steroid hormone,were widely involved in plant growth and response to the environmental factors.BZR1(brassinazole-resistant 1)and BES1(BRI1-EMS-suppressor)were two important transcription factors in the transduction of brassinolide.It had been confirmed in Arabidopsis thaliana that Brassinosteroids could regulate the biosynthesis of Glucosinolates in plants.Sulforaphane and glucosinolate were the most abundant in broccoli,and it was not clear so far whether BZR1 and BES1 transcription factors affect their synthesis in Broccoli and how they were affected.In this study,BZR1 and BES1 genes were cloned from Broccoli,and their expression patterns and functions were further analyzed.The main results showed that:(1)The specific primers were designed according to the nucleotide sequence of BZR1 and BES1 transcription factors of the same species with Broccoli(Brassica),and amplified by RT-PCR from the leaves' cDNA from the Broccoli cultivar "Fuqing No.1" with a high level of sulforaphane.Then the homologous sequences of transcription factors BZR1 and BES1 in the transduction of brassinosteroid were obtained,which were 993bp and 101 lbp in length,encoding 330 and 336 amino acids respectively,and named BoBZRl and BoBESl.Bioinformatics analysis revealed that the nucleotide sequences of the two genes cloned were highly similar to those of other species registered on NCBI and contained typical N-terminal domains and C-terminal transcriptional activation domains,and that they were identified as Broccoli BoBZR1 and BoBESl genes.(2)The subcellular localization vectors pEGAD-BoBZRl and pEGAD-BoBES 1 of BZR1 and BES1 genes were constructed respectively.The results showed that BZR1 and BES1 proteins were located in almost whole cells.After exogenous spraying of BR,all the fusion proteins were concentrated in the nucleus,indicating that BZR1 and BES1 genes were stimulated by BR,and were transported to the nucleus by signal transduction to express the biological function.(3)The qRT-PCR analysis showed that the expression levels of BoBZRl and BoBES1 genes were the highest in roots,the lowest in the flowering cells,and were almost undetectable in seeds.When the leaves were subjected to exogenous spraying of MeJA with a concentration of 500?M,SA with a concentration of 5 mM and BR with a concentration of 3?M,the expression of BoBZR1 and BoBESl genes in Broccoli reached the maximum at the point of 24 h,8 h and 24 h respectively.After 48h of treatment,the gene expression of the group sprayed by BR was slightly decreased,while the other groups tend to initial state,indicating that brassinolide was a highly efficient plant hormones,even a trace can play a sustained role.(4)The plant overexpression vectors pBI-BoBZR1 and pBI-BoBES1,which were driven by the 35S promoter to BoBZRl and BoBES1 genes,were constructed and transformed into wild type Arabidopsisthaliana Col-0.The phenotypic analysis of the transgenic Arabidopsis thaliana homozygote and the detection of the glucosinolate content showed that the overexpression of the two genes showed that the phenotype of the plants were dwarfed and the growth retardation was affected.Specially the overexpression of the BoBESl gene was more affected,and showed smaller and curly leaves.Overexpression of BoBZRl and BoBESl genes reduced the short-chain aliphatic glucosinolates(3MSOP,4MSOB,5MSOP,4MTB)and long chain aliphatic glucosinolates(8MSOO)in Arabidopsis thaliana compared with wild-type Arabidopsis thaliana Col-0,in which the overexpression of BoBES1 gene significantly reduced the content of glucosinolate in Arabidopsis thaliana.The results showed that the expression of BoBZR1 and BBES1 genes had a significant inhibitory effect on the synthesis of glucosinolate.(5)The interference expression vectors of BoBZR1 and BoBESl genes were constructed and transformed into Broccoli with the above overexpression vectors,110 strains of T0 resistant seedlings were obtained.Among them,the plants with BoBZRl and BoBESl genes interference expression vectors were 28 and 26 stains respectively,and the plants with BoBZRl and BoBESl genes overexpression vectors were all 28 stains.