Cloning And Functional Identification Of BoCYP83B1 From Broccoli(Brassica Oleracea Var. Italica)

Glucosinolates are a branch of amino acid-derived specialized metabolites that are specifically found in the Brassicales order.In Arabidopsis,Trp derived indolic glucosinolates are required for plant defense against a wide range of pathogens and herbivores due to their strong antimicrobial activity and potential signaling function.The biosynthesis and degradation of glucosinolate in the model plant Arabidopsis have been well studied.Broccoli is a common Cruciferous vegetable which contains not only comprehensive nutrients and minerals but also lots of glucosinolates.However,little research on glucosinolates in broccoli has been performed,it is of great significance to identify glucosinolate metabolism pathway in broccoli.CYP83B1 is identified to be an important enzyme in indolic glucosinolate biosynthesis pathway that catalyzes indole-3-acetaldoxime?IAOx?to form 1-aci-nitro-2-indolyl-ethane,which is also a precursor of the phytohormone indole-3-acetic acid?IAA?.cyp83b1 show an apical dominance phenotype,which is thought to arise due to blockage of the IAOx-glucosinolates pathway,resulting in accumulation of IAOx,which is channeled into IAA.CYP83B1 is not only involved in the glucosinolate biosynthesis but also affects biosynthesis of IAA.In this study,based on the previous transcriptome sequencing of broccoli we isolated a CYP83B1 from Brassica oleracea var.italica,which we termed BoCYP83B1.To identify its function and detect its expression profile in response to different hormones and stresses,BoCYP83B1 was overexpressed in Arabidopsis and P_BoCYP83B1::GUS transgenic Arabidopsis expressing?-glucuronidase gene?GUS?controlled by BoCYP83B1 promoter was constructed.Our study laid a foundation for futher explore the role of BoCYP83B1 in glucosinolate metabolism and its function in response to different stresses.The main conclusions of this paper are as follows:1.Cloning of BoCYP83B1By RT-PCR,CYP83B1 was cloned from Brassica oleracea var.italica,we named BoCYP83B1 and get accession number KU559565.2.Construction of 35S::BoCYP83B1 transgenic ArabidopsisTo overexpress BoCYP83B1 expression vector 35S::BoCYP83B1 was constructed and transformed into Arabidopsis.Overexpression of BoCYP83B1 in Arabidopsis resulted in an altered glucosinolate profile including an increased content of aliphatic glucosinolates and the indolic glucosinolates was comparable to the wild type plant.And 35S::BoCYP83B1 transgenic Arabidopsis showed an early flowering phenotype.3.Spatial expression pattern of BoCYP83B1By expressing the reporter gene GUS under the control of the BoCYP83B1 promoter in Arabidopsis,we observed that BoCYP83B1 expressed in the vascular tissue of cotyledon,leaves,roots and hypocotyl throughout the whole plant.In the mature plant,GUS signal was clearly observed in the petioles,flower stalks and leaf veins,sepals,the filaments of the stamen,the style of the pistil and young carpels.4.Expression pattern of BoCYP83B1 in response to different hormones and stressesWe analyzed the spatial expression pattern of BoCYP83B1 in response to several hormones and stresses by the reporter gene GUS and realtime RT-PCR.It was strongly induced by methyl jasmonate,1-amino-1-cyclopropanecarboxylic acid?precursor of ethylene?,salicylic acid?SA?,gibberellin acid and IAA.Mannitol,NaCl,UV and Flagelin 22 significantly up-regulated BoCYP83B1 expression.BoCYP83B1 was slightly enhanced by a high or low temperature.The response of BoCYP83B1 to SA,NaCl and wounding showed tissue specificity.