Functional Study Of BnaNIP5;1s In Boron Efficient Uptake And Distribution In Brassica Napus

Boron(B)is an essential micronutrient for higher plant growth and development,which plays important roles in cell wall structure.B deficiency significantly inhibits plant growth,flowering and fruiting setting.Oilseed rape(Brassica napus L.;B.napus)is an important oil crop in China,which is widely planted in the Yangtze River Basin.However,the soil available B in the B.napus production area is low,and most of the soil suffered B deficiency or severe B deficiency.B deficiency often decreases the seed yield and quality of B.napus.A series of physiological and biochemical responses in plants occur to adapt to the fluctuations of external boron concentration.Previous studies have shown At NIP5;1,as an aquaporin,plays an important role in B uptake in roots of Arabidopsis.In this study,we firstly conducted the genome wide study of aquaporin in B.napus(BnaAQPs)and their transcription responses to low B.Then we compared the B uptake efficiency of BnaNIP5;1s between B-efficient cultivar(Qingyou 10,QY10)and B-inefficient cultivar(Westar10,W10),and among different BnaNIP5;1 member of W10.Finally,we analyzed the function of BnaNIP5;1s in B uptake and distribution in B.napus.The main results are as follows.1.Differences in the gene expression of BnaAQPs in response to low B in B.napusBased on the genomic database of B.napus,121 full-length members of AQP gene family were identified by bioinformatics analysis.These members were divided into four subfamilies: BnaPIPs,BnaTIPs,BnaSIPs and BnaNIPs.Generally,the gene expression levels of BnaPIPs and BnaTIPs subfamily were higher than that of other subfamilies,and most of them were down-regulated at low B.The gene expression levels of BnaSIPs and BnaNIPs subfamily were relatively low,and only a few genes had relatively high expression levels.The gene expression of BnaNIP5;1s were significantly up-regulated at low B.Among of them,the gene expression of BnaA07.NIP5;1c and BnaC06.NIP5;1c were almost undetectable in roots of B.napus.BnaA02.NIP5;1a,BnaC02.NIP5;1a,BnaA03.NIP5;1b and BnaC03.NIP5;1b were the gene members with high expression abundance,and they were considered to be the key gene members for B uptake in B.napus at low B.2.Differences in BnaNIP5;1s gene sequence and B uptake ability in B.napusThe CDS sequences of all BnaNIP5;1s members in B-efficient cultivar(QY10)and Binefficient cultivar(W10)were isolated and cloned.Sequence analysis results indicated that the CDS sequences were different between BnaA03.NIP5;1 and BnaC06.NIP5;1,however,only the predicted protein sequences of BnaC06.NIP5;1 showed differences between QY10 and W10.Except for two amino acid difference in BnaC06.NIP5;1 between QY10 and W10,QY10 had an additional 24 amino acid insertion variation.The transient and the stable expression of 35S-BnaNIP5;1s-GFP in tobacco and Arabidopsis thaliana both showed that BnaNIP5;1s were cell membrane localization proteins.The results of heterologous expression of BnaC06.NIP5;1 of QY10 and W10 in yeast and overexpression of them in nip5;1 both indicated that BnaC06 Q.NIP5;1 have lost the ability to absorb and transport B because of the 24 amino acids insertion.The ar/R selective filter region(AIGR,AIAR and ANAR)and TPG repeat sequences region(TPGTPG and KPGTPR)were the important differential sequence regions among BnaNIP5;1s proteins.Both heterologous expression of BnaNIP5;1s in yeast and their complementary experiment in Arabidopsis proved that there were significant differences in B absorption and transport activities among BnaNIP5;1s.The B absorption activity order were as follow.BnaC02.NIP5;1 > BnaA02.NIP5;1 > BnaA03.NIP5;1 and BnaC03.NIP5;1 > BnaA07.NIP5;1 and BnaC06 W.NIP5;1.3.BnaNIP5;1s participates in the absorption of B in roots and the distribution of B in shoots in B.napusRT-PCR results indicated that the gene expression patterns of BnaA02.NIP5;1,BnaC02.NIP5;1,BnaA03.NIP5;1 and BnaC03.NIP5;1 in B.napus cultivar QY10 were similar.They were expressed in root,node and petiole of shoot,and they were all up-regulated in roots and shoots at low B.The tissue location of Pro BnaC02.NIP5;1-GUS was consistent with the gene expression pattern.The expression pattern of NIP5;1 in root of Arabidopsis and in both root and shoot of B.napus were significantly different.The growth of the BnaNIP5;1-RNAi lines was significantly inhibited at low B.The gene expression of BnaNIP5;1s in BnaNIP5;1-RNAi lines were lower than that in QY10 under B deficiency,which resulted in the decrease of B absorption,translocation and distribution in BnaNIP5;1-RNAi lines.These indicated that BnaNIP5;1s had significant roles in the regulation of B homeostasis in B.napus.In conclusion,this study revealed that there were significant differences in transcriptional and coding levels among different gene members of BnaNIP5;1s,BnaA02.NIP5;1,BnaC02.NIP5;1,BnaA03.NIP5;1 and BnaC03.NIP5;1 were members with high expression and high boric acid transport activity;At the same time,it reveals that BnaNIP5;1s genes are involved in the absorption,transportation and distribution of boron in Brassica napus.