Cloning And Functional Characterization Of Drought Tolerance Related Gene BnBiP In Rapeseed(Brassica Napus L.)

Drought, chilling and salinity are major abiotic stresses that seriously affect crop production and quality. Drought, which is the most general abiotic stress, limits the planting area. Therefore, breeding for drought tolerance in crops is an important subject. In this research the BnBiP gene was isolated from Brassica napus L., then the gene was over-expressed in Brassica napus L. by Agrobacterium-mediated transformation, and the function of this gene was analyzed. Furthermore, the drought tolerance mechanism of BnBiP is studied. The main results are as follows:1. According to the known soyBiPD (Accession no. AF031241) cDNA sequence, a sequence about763bp was amplified from mRNA of Brassica napus L. Then the complete cDNA sequence of BnBiP was obtained by Gene RACE. The major open reading frame of BnBiP transcript was1991bp, encoding a650amino acid protein. The structure and functional domain was predicted by SMART, the result showed that BnBiP is highly conserved in plants, and encode3Hsp70conserved domains.2. The BnBip gene was inserted into the binary vector pBI121, the construct was transformed into Brassica napus L. through Agrobacterium-mediated transformation. As a result,21transformants with kanamycin-resistance have been obtained. PCR analysis confirmed the BnBiP gene was successfully inserted into the gemome of Brassica napus L. RT-PCR and Real-time PCR analysis showed that16To transgenic plants were over-expressed.3. The E3ubiquitin ligase gene SDIR is involved in abscisic acid (ABA)-related stress signal transduction, and Over-expression of SDIR1in Arabidopsis thalianais leads to drought tolerance (Zhang et al;2007). Yeast two-hybrid result shows that the interaction between BnBiP and SDIR do not exist. It demonstrated that BnBiP do not involve in the pathway directed by SDIR.4. Tunicamycin inhibits glycosylation of N-linked glycoproteins and, as a consequence, promotes the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which lead to ER stress. The BnBiP over-expressed To transgenic seeds had a higher germination rate than the wide-type in the presence of5μg/ml tunicamycin, and BnBiP overexpressed T1transgenic plants had higher tunicamycin tolerance than control. These results showed that BnBiP as a chaperone in endoplasmic reticulum may confer tolerance to translational inhibition in response to ER stress. 5. RT-PCR and real-time PCR results show that the expression of BnBiP gene could be induced in leaves by drought stress and the expression level significantly increased after stress for1to2hours. The plants over-expressed BnBiP gene significantly enhanced drought tolerance when treated with20%PEG-6000(w/v) for12h or without irrigating for two weeks.6. The expression of AtNRP and GmNAC6(NRP-mediated cell death signaling) in the over-expressed BnBiP transgenic plants was delayed when treated with20%PEG-6000. The result showed that BnBiP may negatively regulate the transduction of the cell death signaling.7. Compared with wide-type, the transgenic plants had the lower water loss rate and MDA content, but higher relative water content and SOD activity. The result indicated that over-expressed the BnBiP gene may improve plant stress tolerance.