Cloning And Expression Analysis Of BnNIP5;1s Of Oilseed Rape (Brassica Napus) With Different Boron Efficiency

Boron (B) is an essential microelement for plant growth and development. Oilseed rape (Brassica napus) is one of the main oil crops in China and showed sensitive to boron deficiency. Six homologous genes of NIP5;1in in oilseed rape (Brassica napus L.) were cloned with homologous gene sequence method and their expression in the shoot and root in B-efficient and B-inefficient cultivars were investigated. The main results are as follow:1. Six homologous genes of NIP5;1were isolated in B-efficient cultivar ’Qingyou10’and B-inefficient cultivar’Westar10’, respectively, and nominated as BnNIP5;1-1a, BnNIP5;1-1c, BnNIP5;1-3a, BnNIP5;1-2a, BnNIP5;1-2c and BnNIP5;1-3c (BnNIP5;1s). Sequence comparative analysis showed that the difference in the sequence between these BnNIP5;1s were located from-1to-100bp in the upstream of the translation initiation site and the first exon. The similarity of each BnNIP5;ls between Qingyou10and Westar10ranged from91.21%to99.99%. Bioinformatics analysis showed that there was no difference in amino acid sequence for each BnNIP5;1s between QingyoulO and westar10. These BnNIP5;1s could be divided into two groups:one group is similar to the homologue genes of NIP5;1located in Brassica rapa A02chromosome and Brassica olerecea C02chromosome, respectively; another group is similar to the homologue genes of NIP5;1located in Brassica rapa A03chromosome and Brassica olerecea C03chromosome, respectively, based on the cluster analysis and neighbor join tree of the homologous NIP5;1s in Brassica crops as well as Arabidopsis thaliana. There existed the functional regions located in5’UTR in each BnNIP5;1s. The sequence of the functional regions between Brassica napus and Arabidopsis were the same, which control the expression of NIP5;1in Arabidopsis.2. Semi-quantitative RT-PCR were conducted to investigate the expression of BnNIP5;1s in the shoot and root of Qingyou10and Westar10. Results showed that the increasing of expression level of BnNIP5;1s were only detected in the root, while very low in the shoot in both cultivars at low B; moreover, the expression level of BnNIP5;1s in the root of QingyoulO showed significantly higher than that of Westar10at both low and high B. BnNIP5;1s may play an important role in the B uptake of oilseed rape. WestarlO showed more quick responses to low B but shorter holdtime in the expression level of BnNIP5;1s under this condition than Qingyou10. The expression level of BnNIPS;1s showed firstly increased then decreased, and then increased again and decreased again in both QingyoulO and Westar10. This indicated that the plants need maintain an appropriate B level, and when it suffered B deficiency, the expression of BnNIP5;1s in the root increased and absorb B from the growth medium to make the B content reach the appropriate B content, then the expression of BnNIP5;1s decreased and B uptake decreased. This process would happen repeatedly for the duration of B deficiency.3. Real-time fluorescence quantitative PCR was used to conduct expression analysis of each BnNIP5;1s in the root. Results showed that the expression level of BnNIP5;1-1a was higher than other BnNIP5;1s under low B stress during all the sampling time, which indicated it may play a major role in response to low B among these BnNIP5;1s; the expression level of BnNIP5;1-3c was the lowest among these BnNIP5;1s under low B stress during all the sampling time, indicating that it could not be induced by B deficiency and contribute little to B deficiency tolerance. All the BnNIP5;1s except for BnNIP5;1-1a showed significant differences in the expression level at different sampling time under low B stress, which indicated that these genes should play different roles at different time under low B. Genes with high expression levels including BnNIP5;1-1a, BnNIP5;1-3a, BnNIP5;1-2a and BnNIP5;1-2c had the same expression pattern as the total BnNIP5;1s obtained from semi-quantitative RT-PCR, that is, the expression of BnNIP5;1s in the root increased firstly, then decreased, and then repeated the process for the duration of B deficiency. These results indicated that the expression level of BnNIP5;1s were regulated by the B concentration in the plant instead of the B content of the growth media.