Study On The Function Mechanism Of Boron Efficient Gene BnaA3.NIP5;1 In Brassica Napus

Boron（B）is one of the essential micronutrients for all higher plant.B deficiency is a worldwide agricultural production problem that has been reported in the field for at least 132 crops from 80 countries.Rapeseed（Brassica napus）is third-largest source of vegetable oil and second-largest source of protein meal in the world,but its production is critically dependent on B supplies.Application of B fertilizer can alleviate B-deficiency problems,but borate rock is a non-renewable mineral resource.Genetic improvement of B efficiency of crops is a promising and cost-efficient strategy in B-deficient regions.Previous studies have indicated that natural rapeseed varieties significantly vary in their tolerance to low-B conditions.However,the genetic basis of low-B tolerance in B.napus tolerance remains elusive.In the present study,based on the identification of Bna A3.NIP5;1 as the candicate gene for q BEC-A3a,we investigated the molecular mechanism of Bna A3.NIP5;1 regulate boron deficiency tolerance in B.napus,and then we analyzed the function diversity of Bna A3.NIP5;1and Bna A2.NIP5;1 in B.napus.The main results are as follows:（1）Gene function of B-efficient gene Bna A3.NIP5;1 in B.napusBna A3.NIP5;1 was identified as a B-efficient candidate gene in B.napus in our previous QTL fine mapping.However,the molecular mechanism through which this gene improves low-B tolerance remains elusive.In the present study,we report genetic variation in Bna A3.NIP5;1 gene,which encodes a boric acid channel,is a key determinant of low-B tolerance in B.napus.Transgenic lines with increased Bna A3.NIP5;1 expression exhibited improved low-B tolerance.Bna A3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap（LRC）cells,where it acquires B for the meristem cells,specifically promotes root growth and therefore leading to high tolerance to B deficiency under B-deficient conditions.（2）Molecular mechanism of Bna A3.NIP5;1 differential expression in B.napusWe first isolated and analsed the promoter of Bna A3.NIP5;1 in the B-efficient cultivar QY10 and B-ineficient cultivar W10.Further analysis with a series of promoeter sequence deletion,recombination and insertion revealed that a CTTTC tandem repeat in the 5’UTR of Bna A3.NIP5;1 altered the expression level of the gene,which is tightly associated with B.napus low-B tolerance and plant growth under B limitation.（3）Nature variation of Bna A3.NIP5;1 and breeding applicationAnalysis with the B-efficient variaties and B-inefficinet variaties from the natural populations revealed that the Bna A3.NIP5;1 in the Bna A3.NIP5;1^Q haplotype was significantly higher than that in the Bna A3.NIP5;1^W haplotype and the increased Bna A3.NIP5;1 expression levels in the representative varieties were positively correlated with primary root length and seed yield under low-B conditions.Field tests with natural populations and near-isogenic lines（NILs）confirmed that the varieties carried Bna A3.NIP5;1^Q allele significantly improved seed yield.Our results provide novel insights into the low-B tolerance of B.napus,and the elite allele of Bna A3.NIP5;1^Q could serve as a direct target for breeding low-B-tolerant cultivars.（4）Function diversity of Bna A3.NIP5;1 and Bna A2.NIP5;1 in B.napusMajor intrinsic protein NIP5;1 is essential for B uptake and plant development under B limitation.In this study,phylogenetic and expression analyses identified five NIP5;1 orthologue genes,but only Bna A3.NIP5;1 and Bna A2.NIP5;1 were mainly expressed in roots of B-efficient veriaty QY10.Bna A3.NIP5;1 located in the distal membrane of LRC cells,Q^{s RNAi} transgenic plants with Bna A3.NIP5;1 expression level specifically decreased,which showed curved leaves,stubby roots and reduced biomass.Pot culture analysis also revealed that Bna A3.NIP5;1 was important for the seed yield of B.napus unde B limitation.Bna A2.NIP5;1 was mainly expressed in the epidermis cells,Q^{m RNAi} transgenic plants with both Bna A3.NIP5;1 and Bna A2.NIP5;1 expression level decreased,which exhibited severer defects,including multiple branches,necrosis in the apical meristem and reduced biomass compared with Q^{s RNAi} transgenic plants.Taken together,we found Bna A3.NIP5;1 specifically localized in the distal part of root tip to promoter root elongation under low-B conditions and Bna A2.NIP5;1 was involved B uptake into roots.Both two genes were important for B.napus growth and development under B limitation,which likely suggest that the coordinate functions of Bna A3.NIP5;1 and Bna A2.NIP5;1 were essential for efficient B uptake as Bna A2.NIP5;1 possibly facilitates B uptake into roots,following the developed root system that Bna A3.NIP5;1 generates.