Study Of Boric Acid Transporter Gene BnBOR1;3s Involved In Boron Response In Brassica Napus

Boron is an essential micronutrient for the growth and development of plants.Boron deficiency is a widespread agricultural problem in the world.Brassica napus(B.napus)has high boron demand and sensitive to boron deficiency.Boron deficiency affects the integrity of the cell wall and the germination of the pollen tube,which thus causes growth inhibition of plant tissues and impairs the reproductive process of floral organs,and finally leads to reduced or no yield.We made progress on identifying the borate transporter gene BnBOR1;1c expressed specifically in tissues,which is involved in boron homeostasis,while there are few studies on constitutive expression genes of BnBOR1;3s.The BnBOR1;3s thus are candidates.We investigated the phylogenetic relationship,boron transport capacity,response to boron stress and localization of BnBOR1;3s.Further genetic analysis proved that BnBOR1;3s are involved in the transport and regulation of boron in Brassica napus.The main results are as follows:1 Analysis of BOR1 family in Brassica napusWe analyzed the physicochemical properties and characteristics of six BnBOR1 s,BnBOR1 s had high homology with At BOR1,the identity was more than 89%.Then,according to the evolutionary relationship of BOR1 in Brassica napus,Brassica rapa,Brassica oleracea,maize,wheat,rice and barley,we found out the six BnBOR1 s are from Brassica rapa and Brassica oleracea.Finally,we found the protein identity of BnBOR1 s and At BOR1 was more than 90%.2 Analysis of boron efflux transport capacity of BnBOR1;3sWe validated boron efflux activity of BnBOR1 s by using yeast expressing target genes.It was showed all BnBOR1 s had the capacity to transport boron,while BnBOR1;3a gathered less intracellular concentrations of boron and got more boron export.Then,We found serine 430 of BnBOR1;3a is different from other BnBOR1 s,according to the prediction of protein tertiary structure,the mutant of serine 430 changed the conformation of BnBOR1;3a.Otherwise,site-directed mutagenesis of serine 430 and serine 660 showed,they are critical amino acids to maintain a high boron export capacity of BnBOR1;3a.3 Expression pattern and localization of BnBOR1;3sIn QY10,BnBOR1;3s expressed in root,hypocotyl and node.They were not induced by boron deficiency,and their expression in the root were significantly higher than that in the shoot.in Arabidopsis thaliana,The report gene GFP was used to investigate the location of BnBOR1;3s.BnBOR1;3s were strongly expressed on the plasma membrane of the root cell.Otherwise,BnBOR1;3c-GFP protein level was not degraded under a high boron condition.4 Gene function of BnBOR1;3sUnder low boron stress,in Brassica napus,the knockout lines of BnBOR1;3c developed more severe symptoms of boron deficiency than wild type.The growth of the main root was blocked,the root tip was mallet-shaped and could not stretch normally,the leaves were thickened and darkened,and the leaf margin was obviously curled and shrunk;in Arabidopsis thaliana,We found that the overexpression lines of BnBOR1;3s significantly increase fresh weight and promote root length compared with the mutant.These results proved BnBOR1;3s have a great influence on boron homeostasis in Brassica napus.In conclusion,BnBOR1;3s are constitutive genes and strongly expressed in the root of Brassica napus;BnBOR1;3s protein are expressed on the plasma membrane and not degraded under a high boron condition;Genetic analysis showed BnBOR1;3s are involved in boron homeostasis in Brassica napus.