Study On Analysis Of BnBORs Family Members And Function Of BnaC4.BOR1;1c In Brassica Napus

Boron(B)is one of the essential micronutrients for plants and boron deficiency affects the quality and quantity of agricultural production.Oilseed rape(Brassica napus)is one of the major oil crops in China,which accounts for more than 80% production of rapeseed.But the production of oilseed rape suffers a loss of B deficient in the main production areas.The B uptake,transport,utilization and distribution was variations in different genetypes of Brassica napus.Previous study found the relationship between the B transporter and the different B efficiency Brassica napus.This research mainly introduced the function of Bna C4.BOR1;1c,which played a role in the inflorescence growth and reproduction in Brassica napus and the different promoter regions regulate B transport between the high efficiency cultivar and the low efficiency culticar.The main results are as follows:1.Using the sequenced database of rapeseed genome,20 BOR members of Brassica napus were identified.And,their physical and chemical properties,phylogenetic tree,gene structure,chromosomal location and protein structure were analyzed.According the high homology of Arabidopsis thaliana BOR members,there were 6 groups B transporter genes in Brassica napus.These genes had the similar gene structure,conservative HCO3-motif and extensive distribution on the chromosome.We explored their expression profile in different B efficiency varieties of Brassica napus under different B conditions,and results showed distinct expression profile,which may indicate various gene function among the BOR members.2.Two report gene GUS and GFP were used to investigate the subcellular and histological localization of Bna C4.BOR1;1c.Distinct GUS staining was observed in the nodes at the base of the rosette leaves and the mature root zone at the seedling stage but not in the leaves and root tips.Investigation of Bna C4.BOR1;1c expression in inflorescences showed that the GUS was distinctly expressed in stem nodal regions and floral buds.The subcellular results showed the GFP signal was observed in the entire root,including meristematic,transition and mature zones especially around vascular tissues.Therefore,Bna C4.BOR1;1c is a PM-localized protein as same as At BOR1.3.We performed a functional complementation test by overexpressing Bna C4.BOR1;1c in Arabidopsis ecotype Col-0 and the mutants bor1-1.Three independent lines were used for phenotypic investigation under low B(0.3 ?M)and normal B(30 ?M)conditions.The transgenic plants with much higher expression of Bna C4.BOR1;1c displayed stronger tolerance to low B stress,and their shoot fresh weights and primary root length were significantly higher than Col-0 and bor1-1 mutants.In addition,overexpressing Bna C4.BOR1;1c in bor1-1 significantly increased the B concentration in shoot.These results demonstrated that overexpression of Bna C4.BOR1;1c in bor1-1 rescued the growth of bor1-1 mutants under B deficiency,suggesting that Bna C4.BOR1;1c has a similar function to At BOR1.By contrast,overexpression of Bna C4.BOR1;1c in nip5;1-1 mutants did not improve the tolerance to low B stress.4.To elucidate the effects of Bna C4.BOR1;1c on plant growth and B transport,we established rapeseed Bna C4.BOR1;1c knockdown lines by RNAi technology.Under vegetative and reproductive stage,we found that Bna C4.BOR1;1c transports B into the xylem for translocation to the shoots,which is critical for the development and fertility of inflorescences under B limitation in allotetraploid rapeseed.Compared with that of QY10,under low B condition,the growth of the RNAi lines displayed a range of typical B deficiency symptoms,including large numbers of abnormal flowers with exposed stigma and dried-up and dropped floral buds and especially notable at the main inflorescence.The flower organs of the QY 10 plants developed normally,with six strong stamens and normal stigmas,but the knockdown lines produced small and malformed stamens and atrophic pistils with a browning epidermis below the stigma.The B concentrations in flowers of the knockdown lines were significantly lower than that of QY 10 plants.All these resulted finally in significantly lower seed yield than that of QY 10 plants.The pistils of the RNAi plants with a brown epidermis were stained red,indicating the presence of higher levels of lignified tissues in RNAi lines.Histological analysis showed that the mastoid cells and parenchyma cells on the stigma were not present in the RNAiplants.The SEM analysis of the developing stigma revealed that the central region of the stigma surface in RNAi lines was sunken,and large amounts of stigma papilla cells were disintegrated.Moreover,broken anthers and immature 3-aperture pollen with more cell debris were also observed in the RNAi lines.TEM analysis of the stylar canal below the stigma showed that the cells of RNAi tissues were smaller and more compact,indicating the cell expansion was repressed in RNAi tissues.Furthermore,the cell walls swelled,and the space between cells was compressed in RNAi lines.5.According the expression of Bna C4.BOR1;1c in QY 10 and Westar 10 in response to B limitation,Bna C4.BOR1;1c was up-regulated distinctly in the case of B deficiency and the expression was higher in QY10 than Westar 10.In the B gradient experiment,QY10 plants showed higher B concentration and better growth than those of Westar 10 plants.The differential expression of Bna C4.BOR1;1c may contribute to the high B efficiency in QY 10.We isolated and assessed the coding sequences and promoter of Bna C4.BOR1;1c from the two cultivars using BLAST and found no difference in gene sequence and two indels in the promoter regions.One is an insertion of 27 bp at-478 bp to-451 bp in the QY 10 promoter.Another is an insertion of two TTC repeats at-388 bp to-382 bp of the Westar 10 promoter.Furthermore,four cis-acting elements,YACT,GATA BOX,IBOXCORE,and ROOTMOTIF TAPOX ?,were identified in the 27 bp insertion in the QY 10 promoter region using the PLACE website.These results indicated that the 27 bp insertion in the Bna C4.BOR1;1c promoter may be critical for the differential expressions between QY 10 and Westar 10 plants.