Cloning And Functional Characterization Of Drought Resistance And Salt Tolerance Related Genes Bnsdir1and Bnrab7in Rapeseed(Brassica Napus L.)

Abiotic stresses are the major damage to agriculture in the world. Under abiotic stress, plants could sense and transduce the stress signals then initiate a series of functional genes related to stress tolerance. Meanwhile, physiochemical reactions can be activated to alleviate the damage caused by the stress.In present study, BnSDIRl and BnRab7genes were isolated from Brassica napus. BnSDIR1gene encodes an E3containing a RING motif in its C-terminal region, BnRab7is a small GTP-binding protein derived from the Rab protein family. Based on the evaluation of drought resistance and salt tolerance, we found that the BnSDIR1and BnRab7were candidate genes for developing salinity and dehydration tolerance in plant.The main results were as follows:1. Isolation and characterization of BnSDIRl(1) BnSDIR1gene was cloned by the homologous sequence method. It contained two putative transmembrane domains at the N-terminus and a full RING finger at the C-terminus.(2) The expression analysis of RT-PCR and Real-time PCR in TO and T1plants demonstrated that higher expression abundance of BnSDIR1in the transgenic plants induced by drought treatment after0.5-1h.(3) The transgenic plants of overexpression BnSDIR1showed hypersensitivity to10mg/L ABA at the germination, and the germination rate and hypocotyl length were evidently less than the control. Under the stress of20%PEG-6000, the analysis on the transcription of BnSDIRl and other genes that were known as upstream genes in the ABA signaling pathway, ABA-responsive bZIP transcription factors and other genes responsed to stress shed lights on that BnSDIRl gene had a possible functional role in ABA transduction pathway.(4) Under drought stress (20%PEG-6000treatment for5-6h/withdraw water for15d), the BnSDIRl transgenic lines enhanced drought resistance. Also, we found that transgenic lines showed salt hypersensitivity in germination, but increased salt tolerance after seedling stage. Based on the analysis of the physiological and biochemical indexes such as relative water content (RWC), malondialdehyde (MDA), superoxide dismutase (SOD), we found the significant differences between the transgenic lines and control.2. Isolation and characterization of BnRab7(1) BnRab7gene was cloned by the homologous sequence method from Brassica napus. The BnRab7protein showed the presence of functionally important domains involved in GTP binding and hydrolysis domains designated as G1and G3-G5, the C terminal Cys residues for membrane attachment, the G2domain for effector binding. The alignment of several Rab7proteins indicated Rab7was not conserved in the plant kingdom, but high homology existed even with mammals and yeast.(2) At the four or five leaf stage, the transgenic lines and the control were treated with200mmol/L NaCl, and the samples were taken at different times in order to extract RNA. The RT-PCR analysis revealed the expression of BnRab7upregulated in NaCl treatment, and the increased expression appeared after1h.(3) The BnRab7transgenic lines partly in liquid culture were treated with200mmol/L NaCl and the others were treated with600mmol/L NaCl in the soil, the plants improved salt tolerance compared to the control. Based on the evaluation of the physiological and biochemical indexes such as relative water content, malondialdehyde (MDA), superoxide dismutase (SOD) indicated the transgenic lines were better than the control.