| Soil salinization has been a main barrier in protected vegetable production and seriously hampered the sustainability of production.Cucumber?Cucumis sativus L.?is widely planted in field and facilities,but soil secondary salinization has restricted cucumber production.The main stress anion is nitrate.Plants assimilate nitrogen element through nitrate assimilation pathways in the form of NO3-and NH4+.The assimilation of nitrate is a tightly regulated process in which NR and NiR play a major regulatory role.As a key enzyme and a rate limiting enzyme in the process of nitrate nitrogen assimilation,NR plays a key role in plant nitrogen metabolism.NR also can synthase NO in plants.NO,in a manner,can improve plant resistance to stress and alleviate adversity stress.Therefore,to investigate the effects of CsNR on antioxidant system and nitrogen metabolism of plant seedlings under NO3-stress play an important theoretical and practical significance.The influence of CsNR transgenic plants which were obtained by agrobacterium tumefaciens infection,on oxidation system,expression characteristics of nitrogen metabolism and molecular regulation mechanism on endogenous NO under NO3-stress in cucumber is studied.The main results are as follows:1.RT-PCR primers were designed according to the published cucumber nitrate reductase gene sequence?login number NM001280767?in GeneBank,and the recovered PCR products were connected to the pMD18-T vector.The full-length CDS was 2952 bp and the fragment was 360 bp.The sense expressing vector named pROK?-CsNR?+?was digested by the fragment CDS and pROKII.The antisense expressing vector named pROK?-CsNR?-?was digested by the fragment and pROKII.The homology alignment analysis indicates that the protein encoded by CsNR was in high similarity with NR-encoding protein in Cucumis melo,Nicotiana tabacum,Arabidopsis thaliana and Brassica campestris.The homology with amino acid sequences of CsNR compared with Cucumis melo is the highest by 98.25% and Nicotiana tabacum is 78.67%.The homology with amino acid sequences of antisense compared with Nicotiana tabacum is the highest by 90.83%.2.In this study,the genetic transformation test of CsNR gene was carried out by ovaryinjection and agrobacterium infection method,and identified by PCR.The transformation of genetic transformation in cucumber plants is unsuccessful.PROK II-CsNR?+?and pROK IICs NR?-?were successfully transferred into tobacco using agrobacterium tumefaciens method.And T0 transgenic tobacco is detected by PCR showed that the target gene has been successfully transferred into the regenerated plants,and its transformation rate is 92%.3.We investigated the effect of nitrate stress on antioxidant system in transgenic tobacco.It is found that NO3-stress can reduce the activity of POD and CAT,so it accumulate H2O2,and it do harm to the plant.Compared with wild type,CsNR?+?tobacco maintained a high level of CAT activity and can scaveng H2O2 and protect plants from harm.The activities of SOD and GR of CsNR?-?transgenic tobacco were significantly increased under NO3-stress treatment.This can effectively remove superoxide radicals in cells due to nitrate stress,reduce accumulation of AsA,and reduce physiological damage caused by plants under stress.In conclusion,CsNR?+?transgenic tobacco alleviated NO3-stress by improving antioxidant enzyme activities and reducing membrane lipid peroxidation.4.We investigate the effects of nitrate stress on nitrogen metabolism in transgenic tobacco seedlings.We find that CsNR?+?transgenic tobacco can increase the activity of NR,NiR,GOGAT,GS,GDH,and its gene expression level under the stress of NO3-.CsNR?-?is the opposite.The change of gene expression is basically consistent with enzyme activity.5.We investigate the effects of nitrate stress on NO in transgenic tobacco plants,and find under the stress of NO3-,NO produce by tobacco seedlings regulates NR content in plants by regulating the activity of NO,and enhances the ability to resist stress. |
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