Mechanism Of Tobacco Over-expressed The Superoxide Dismutase (SOD) And Catalase (CAT) Genes From Dambo Black Soybean Response To Aluminum Stress

Aluminum toxicity is one of the main factors limiting plant growth in acid soils.The traditional way to change acid soil is to use lime and complexing agent to improve the soil,however the effect is not ideal.Therefore,we studied the physiological and biochemical mechanism of aluminum tolerance in plants,and used genetic engineering technology to cultivate transgenic aluminum-tolerant plants to improve aluminum toxicity and sustainable productivity in acid-resistant soils.In this study,aluminum-resistant Dambo black soybean was used as experimental material and its GmSOD and Gm CAT genes were cloned.The prokaryotic expression vector of GmSOD gene was constructed,and the purified protein was induced and analyzed.The plant overexpression vector p K-35S-GmSOD was constructed and transformed into tobacco to obtain overexpressing GmSOD tobacco.The relative root growth(RRG),the content of hydrogen peroxide(H₂O₂),the gene expression and activity of antioxidant enzyme system and other physiological and biochemical indexes related to aluminum stress in transgenic GmSOD tobacco roots under aluminum stress were analyzed,and then the relationship between these physiological indexes was analyzed to reveal the physiological and biochemical mechanism of tobacco response to aluminum stress.Meanwhile,transgenic GmSOD tobacco was used as material.The Gm CAT gene was transformed into transgenic GmSOD tobacco to obtain overexpressed GmSOD and Gm CAT double transgenic tobacco callus.In addition,the effects of double transgenic genes on the content of endogenous H₂O₂in tobacco callus and the regulation of H₂O₂on the growth and development of tobacco were discussed.The main results are as follows:1 The full length of GmSOD gene was cloned from dambo black soybean root,and the prokaryotic expression vector of GmSOD was successfully constructed.The enzymatic characteristic analysis showed that the optimum temperature of GmSOD was 60?and the thermal stability was good.The optimal p H is 7.5,which is relactively stable in the range of 5.5-9,and the acid-base stability is good.However,enzyme activity is significantly inhibited and easily inactivated when the p H value is less than 5.5 or greater than 9.1m M K⁺,Mg²⁺,Ca²⁺,and Al³⁺activated the enzyme activity and activate capacity is K⁺>Mg²⁺>Ca²⁺>Al³⁺;it inhibited enzyme activity when the metal concentration is 3 m M.3m M Cu²⁺and Mn²⁺inhibited enzyme activity,but the inhibition of Cu²⁺was stronger.Na⁺has little effect on enzyme activity.2 The plant expression vector p K2WG-35S-GmSOD of GmSOD gene was successfully constructed by Gateway technology.The transgenic tobacco of GmSOD gene was obtained by Agrobacterium p MP90 transfection.The content of H₂O₂and MDA in the root tip of wild-type tobacco were significantly higher than that of transgenic tobacco after 7 days of cultivation in 0,50,100,200 and 400?M aluminum solution,which indicated that transgenic tobacco had stronger aluminum resistance than wild-type tobacco.The correlative factors of secreting citric acid in wild-type tobacco and overexpressing GmSOD gene tobacco root tip in response to aluminum stress including the stress time,stress concentration,H₂O₂content,MDA content,the soluble protein content and superoxide dismutase enzyme activity,peroxidase enzyme activity,catalase enzyme activity,ascorbic acid peroxidase enzyme activity,PM H⁺-ATPase were used for factor analysis by SPSS.The results showed that the main two factors affecting the response of tobacco to aluminum stress were"antioxidant capacity"and"aluminum toxicity".Under low aluminum stress(wild type tobacco<100?M,transgenic tobacco<200?M),the"antioxidant capacity"was greater than the"aluminum toxicity"in the tobacco root tip,and the accumulated H₂O₂concentration in tobacco was low(0.35-0.6?mol g^-1FW),which could induce the expression of antioxidant enzymes,improve the activity of antioxidant enzymes and maintain the low H₂O₂content in the root.Low concentration of H₂O₂acts as a signal molecule to promote the phosphorylation of plasma membrane H⁺-ATPase and its combination with 14-3-3 protein to increase the hydrolytic activity of PM H⁺-ATPase and H⁺pump activity in tobacco root tip,and to promote the secretion of citric acid,increase resistance to aluminum toxicity.Moreover,the change of enzyme activity showed a certain time rule.Whether it is wild tobacco or transgenic tobacco,SOD gene increased rapidly and reached the highest level at 24 h.Then,the expression and enzyme activity of CAT,POD and APX genes increased,reaching the highest level at 48 h.Under high aluminum stress(wild-type tobacco>100?M,transgenic tobacco>200?M),the"antioxidant capacity"was less than the"aluminum toxicity"in the tobacco.Producing high concentration of H₂O₂inhibited the phosphorylation of PM H⁺-ATPase,thus inhibiting thier interaction level with 14-3-3 protein,reducing the activity of PM H⁺-ATPase and H⁺pump,and reducing the secretion of citric acid.3 The plant overexpressing vector p K2-35S-Gm CAT of Gm CAT gene,which was successfully constructed by Gateway technology,was transfected into GmSOD tobacco through Agrobacterium p MP90,and the callus transgenic tobacco with double GmSOD and Gm CAT genes was obtained.Callus showed abnormal growth and development,and only divided but did not differentiate into cluster buds.The content of endogenous H₂O₂in tobacco callus was found to be lower than 0.1?M.The callus was transplanted to supplemented exogenous 0.235?M H₂O₂MS culture medium for7 days.The endogenous H₂O₂of callus increased to more than 0.1?M.The callus grew well and the budding rate increased.The expression of Tobacco mitogen-activated protein kinase kinase kinase(NPK1),Ca²⁺/calmodulin-dependent kinases(Ca MK),Glutamate-glyoxylate transaminase(GGAT)and mitogen-activated protein kinase(MAPK)related to growth and development of double transgenic tobacco was analyzed by RT-PCR.The results showed that the expression of these genes increased with the addition of exogenous H₂O₂,which indicated that overexpression of both GmSOD and Gm CAT genes in tobacco resulted in the decomposition rate higher than the production rate of H₂O₂,and the content of H₂O₂was too low(less than 0.03?M g^-1FW),which could not be used as a signal molecule to regulate the normal growth and development of plants.