Research On The Absorption And Metabolism Of Formaldehyde In Tobacco By The Regulation Of 14-3-3 Protein

It is simple,beautiful and environmentally friendly to use ornamental plants to purify formaldehyde(HCHO)pollution in the environment.However,the ability of wild-type plants to absorb and metabolize HCHO is relatively limited.Therefore,it is necessary to study the mechanism of plant absorption and metabolism of HCHO,and to obtain genetic operation strategy for improving plant absorption and metabolism of HCHO.14-3-3 protein is a regulatory protein in plant cells,which is involved in the response of plants to abiotic stresses.Bivalent cations such as Mg2+,as well as polyamines such as spermine and spermidine could bind with 14-3-3 protein,and induce conformational changes of the target protein binding region in 14-3-3 protein,then,they can be involved in the regulation of the interaction between 14-3-3 protein and target proteins in plants.In this study,tobacco as a model plant was used as experimental material,physiological and biochemical methods were used to study the effects on stress response,absorption and metabolism of tobacco leaves under aqueous and gaseous formaldehyde stress with the exogenous application of Mg2+,spermine and spermidine.The tobacco 14-3-3 genes with differental expression in both aqueous and gaseous HCHO sterss was screened,14-3-3 gene transgenic tobacco was constructed by transgenic operation,and the regulatory effect of 14-3-3 protein on tobacco HCHO absorption was verified,the main research results are as follows:1.Detached tobacco leaves were treated in 4 mmol·L-1 aqueous HCHO with the application of Mg2+,spermine and spermidine.The results showed that the exogenous application of Mg2+(5 mmol·L-1),spermine(100μmol·L-1)or spermidine(200 μmol·L-1)can significantly improve the absorption efficiency and metabolic capacity of tobacco leaves in aqueous HCHO.These 14-3-3 protein interaction activator can reduce the degradation of chlorophyll and enhance the activity of antioxidant enzymes under aqueous formaldehyde stress.In this way,the resistance of tobacco to aqueous HCHO was enhanced,and peroxidation damage of tobacco mesophyll cells was reduced,therefore,the absorption of aqueous HCHO by tobacco leaves was increased by 33%～40%.In addition,the exogenous application of Mg2+ and spermine reduced the consumption of endogenous[U-13C]Gluc and[U-13C]Fruc in aqueous HCHO stress,Besides the original metabolite[U-13C]Gln andU-13C]Asn,the new metabolites[4-13C]Glu,[3-13C]Asp,[U-13C]Mal and[U-13C]PEP were also found in metabolic profiling.It is speculated that 14-3-3 protein may enhance the metabolism of tobacco leaves by regulating the activity of enzymes(MDH and GS)involved in the pathway of tobacco HCHO metabolism.2.The roots of tobaccos were treated with Mg2+,and spermine and spermidine for 3 h respectively,and then tobacco plants were treated with gaseous HCHO.The results show that the pretreatment of tobacco roots with Mg2+,spermine aid spermidine could improve HCHO absorptive capacity of tobacco plants in 1,3 and 5 mg m’3 gaseous HCHO,among them,the effect of spermine was the most significant and the absorption efficiency of gaseous HCHO was increased by 24%-37%.Under gaseous HCHO stress,the application of Mg2+,and spermine and spermidine could protect chlorophyll,regulate the activity of antioxidant enzymes,delay the accumulation of MDA and H2O2,and reduce the oxidative damage of tobacco leaves,but it had no effect on the metabolism of gaseous HCHO.3.RT-PCR method was used to analyze the expression of 14-3-3 gene in tobacco leaves under aqueous and gaseous HCHO stress,expression analysis of 14-3-3 genes under HCHO stress displayed that there are only 14-3-3c and 14-3-3g gene showed significant differential expression in the aqueous and gaseous HCHO at the same time.The over expression(pK2·35S-14-3-3)and RNA interference(pK7-35S-14-3-3-I-14-3-3)plant expression vector of differentially expressed genes among tobacco 14-3-3 genes were constructed using Gateway technology,finally,transgenic tobacco with 14-3-3 gene were obtained to verify the regulation of 14-3-3 protein.Further analysis showed that it enhanced the resistance of tobacco to HCHO by the overexpression of 14-3-3c gene(KC1)and 14-3-3g gene(kg1),and the absorption ability of transgenic tobacco to liquid HCHO was increased by 10%and 13.1%,respectively.These results confirmed that 14-3-3 protein was involved in the response to HCHO stress and regulation of HCHO absorption in tobacco.it provides a new strategy for improving the ability of plant to repair HCHO pollution by genetic engineering.