The Study Of SoHb Function And SoGSNOR Expression Of Spinach (Spinacia Oleracea L.) Under Nitrate Stress

The secondary salinization of soil is one of the main obstacles to the vegetable production,which severely restrict the sustainable development of greenhouse vegetables.Studies have shown that the anion in the secondary soil is mainly NO3-.NO is a signaling molecule,which plays an important role in seed germination,growth and biotic or abiotic stress tolerance.Plant non-symbiotic hemoglobin(nsHb)and S-nitrosoglutathione reductase(GSNOR)play an important role in the metabolism of NO.This paper focuses on the function and expression of SoHb and SoGSNOR under nitrate stress.The main results are as follows:1 We investigated the effect of exogenous NO donor sodium nitroprusside(SNP)on the metabolism of reactive oxygen species(ROS)and reactive nitrogen species,(RNS)under nitrate stress in two spinach cultivars(resistant varieties Chaoji,CJ)and salt-sensitive species 'Daye',DY).The results showed that the growth of spinach was inhibited after excess nitrate treatment.The fresh weight and dry weight decreased and the TBARS and H2O2 contents increased.After SNP was added into the nitrate solution,compared with the nitrate treatment,the ROS content decreased,especially in the CJ cultivar.The antioxidant enzyme of SOD,POD,CAT,APX,GR all increased,while The NR activity decreased significantly and the NOS activity increased.The NO content increased after nitrate treatment.When tungstate or L-NAME were added,the NO content decreased.The SNO content increased after nitrate treatment with or without SNP.2.The prokaryotic expression plasmid vector pET32a-SoHbwas constructed and transformed into E.coli BL21 star(DE3)strain for genetic engineering strain.The transformed strain was induced with isopropyl-beta--D-thiogalactoside(IPTG)for expressing fusion protein.SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 38 kDa was highly expressed in E.coli and presented both in the supernatant and the pellet part of E.coli lysates.The pET32a-SoHb strain was more resistant to nitrosative stress than the pET32a empty vector strain.The supemaatant was further purified by Ni2+ NTA affinity chromatography and immunized white mouses as antigen.The polyclonal antibody was obtained and analyzed by western blot.3.The nitrate and SNP tolerance of SoHb overexpressing transgenic Arabidopsis were studied.The results showed that:the overexpression of SoHb resulted in decreased tolerance to nitrate stress,as shown by reduced root length,fresh weight,the maximum photosystem ? quantum ratio of variable to maximum fluorescence,and higher malondialdehyde contents than that of the wild type(WT)plants.The NO levels in SbHb-overexpressing transgenic plants were lower than that of WT plants.The activities and transcription of superoxide dioxidase,and catalase decreased more than that of the WT plants under nitrate stress,suggesting that overexpression of SoHb rendered plants more susceptible to oxidative damage.Expression levels of RD229 RD29A,DREB2A,and P5CS1 decreased after nitrate treatment in SoHb over-expressing plants.Moreover,SoHb-overexpression plants were more tolerant to SNP.The root length and fresh weight were higher than the WT plants.The NO fluorescence was lower than the WT plants after NP treatment.Besides,the flower time of the transgenic plants were earlier than the WT plants,and the transcript level of CO,SOC,FT,FLC,GI were higher than the WT.4.The full length cDNA sequence of SoGSNOR was obtained.Sequence alignment of the predicted amino acid residues with different members of the GSNOR family clearly established that SoGSNOR had high similarity with other GSNOR proteins.The gene and protein of SoGSNOR expression were analyzed under nitrate stress and different exsogenous NOtreatment by real-time PCR and western blot.The results showed that the transcript levels of SoGSNOR were decreased after nitrate treatment.With the addition L-NAME conditions' the expression of SoGSNOR was significantly increased.The prokaryotic expression vector of pET32a-SoGSANOR was constructed and get about 65kD target protein induced by IPTG,the supernatant was further purified by Ni2+ NTA affinity chromatography and immunized white mouses as antigen.The polyclonal antibody was obtained.The overexpressing plant expression vector pRI101-SoGSNOR was constructedand and transformed into tobacco.The transgenic plants were analyzed by genomic DNA PCR.The S-nitrosylation related protein was analyzed by biotin switch method.Six S-nitrosylation proteins about photosynthesis of spinach leaves were obtained.