Functional Identification Of BnTR1 In Drought Resistance And Salt Tolerance Of Brassica Napus

Rapeseed is one of the most important oil crops in China and even in the world.However,in the course of its life,it will encounter a variety of adverse conditions,such as drought,salinity and other abiotic stresses environment,which seriously affect the growth of rapeseed,and lead to a significant reduction in production.E3 ubiquitin ligases play an important role in plant response to abiotic stress.For instance,U-box,SCF and RING-finger composite type E3 ubiquitin ligase involved in the drought stress response pathway.BnTRl encodes an E3 ubiquitin ligase with an RING-finger domain.In this study,drought and salt stress treatment was carried out on the Brassica napus "Yangyou No.9"(Y9)and the BnTR1-overexpressing transgenic plants during germination and seedling stage to identify the function of BnTRl in B.napus response to drought and salt tolerance.The main results of this study are as follows:(1)The PCR and qPCR was used to identify the BnTR1 T0 generation transgenic plants.As a result,we obtained eight BnTRl overexpression lines,and the expression level of BnTRl in the transgenic plants was 2.8-6.7 times as high as that in Y9.(2)We analyzed agronomic traits of Y9 and the T1 generation transgenic plants overexpressing BnTRl,including plant height,1000-grain weight,yield per plant and so on.We found that overexpression of BnTRl had no significant effect on the yield of B.napus.(3)Y9 and the T1 generation transgenic seedlings overexpressing BnTR1 were subjected to the natural drought stress by withholding watering for two weeks,and the physiological indexes of leaves before and after treatment were determined.The results showed that the content of soluble protein and the SOD and POD activity in transgenic seedlings overexpressing BnTRl was significantly higher than that in Y9 under drought stress,and the MDA content in transgenic plants was lower than that in Y9.After germination for 7 days with PEG-6000 treatment,we found that the germination rate,seedling height,and root length of transgenic seedlings overexpressing BnTRl was significantly higher than that of Y9 plants,and transgenic plants accumulated less H2O2 and O2·-in leaves.Transcript abundance analysis showed that several antioxidant enzyme genes was up-regulated in the transgenic plants.(4)The effects of 600 mM NaCl treatment on Y9 and the T1 generation transgenic seedlings overexpressing BnTRl were investigated.The results showed that the growth of transgenic seedlings was superior to that of Y9 plants.The SOD,POD and CAT activities in transgenic plants were higher than that in Y9 plants,while the MDA content in transgenic plants was lower than that in Y9 plants.After 7 d treatment with 150 mM NaCl,it was found that the germination rate,seedling height and root length of transgenic seedlings was higher than that of Y9 plants.The results of DAB and NBT staining showed that the accumulation of H2O2 and O2·-in leaves of transgenic seedlings was less than that of Y9 plants.qPCR analysis revealed that the expression of several antioxidant enzyme genes was increased in transgenic plants.(5)The plant expression vector pMDC83-BnTR1,which was used for subcellular localization,was constructed by restriction enzyme digestion and ligation approach.The expression of BnTR1-GFP fusion protein in cells was detected by fluorescence confocal microscopy.The results showed that the BnTR1 protein was likely to localize on the cytoplasmic membrane and in the cytoplasm.(6)The bait vector pGBKT7-BnTR1,which was used for yeast two hybrid,was constructed.In this study,five potential interacting proteins of BnTR1,including calreticulin 1b,catalase 2 and CIPK9,were obtained by yeast two-hybrid screening.In this study,eight transgenic lines overexpressing BnTR1 were identified by PCR and RT-PCR analysis.Compared with Y9,the drought resistance and salt tolerance of transgenic plants overexpressing BnTR1 was increased.Based on the results of this study,we speculated that BnTR1 could enhance the drought resistance and salt tolerance of B.napus via antioxidant enzyme system to remove ROS and relying on the interaction with its interacting proteins.