Transformation Of Tobacoo And Lotus Japonicus With E.coli HspQ Gene

Lotus corniculatus L.is an excellent perennial forage of the Lotus family,which has good application value in the karst mountainous areas,and drought and high salt stress conditions are one of the main factors limiting the growth of Lotus corniculatus..In some tobacco districts in China,there are often periodic water shortages that have severely affected their production and quality.Therefore,the research on improving the drought resistance of tobacco has been paid more and more attention.Heat shock proteins(HSPs)are a class of protective proteins produced by organisms under stress conditions.They mainly protect the cells through molecular chaperone mechanisms and increase plant tolerance to stress,and are closely related to stress responses to stress.Related.In this thesis,the gene encoding HspQ of E.coli heat shock protein was genetically transformed into tobacco and Lotus root,and the effects of the expression of the gene in tobacco and Lotus root on the growth and development of tolerance plants,tolerance to stress and their physiological and biochemical mechanisms were studied.To explore the feasibility of using HspQ gene to create a new germplasm of stress-resistant grasses,and to lay a foundation for molecular breeding of stress-resistance herbage.The results obtained are as follows:1 HspQ Gene Biological Function Prediction AnalysisBioinformatics analysis of the E.coli HspQ gene.The results showed that the encoded protein contained 105 amino acids and was a kind of hydrophilic protein.There were 28 phosphorylation sites in the protein,including 17 serine sites,7 threonine sites and 4 tyrosine residues.The signal peptide may be present in the protein of interest and there is no transmembrane domain.The analysis of the secondary and tertiary structures showed that the protein encoded by HspQ is mainly composed of irregular coils and alpha helices.2 Genetic Transformation of Tobacco and Lotus Roots with HspQ GeneThe 35 S promoter was used to drive the expression of E.coli HspQ gene and gus::npt as the plant expression vector pSHHspQ of reporter gene and resistance selection marker gene.Agrobacterium-mediated genetic transformation method and vacuum permeation transient transformation method were used to transform plant tobacco and target plant Lotus corniculatum.GUS histochemical staining and genomic PCR identification indicated that 15 transgenic tobacco lines and 20 transgenic lines were obtained.Lotus root.3 Effect of HspQ Gene Expression on Drought Resistance of TobaccoThe transgenic tobacco was treated with drought stress with 10% PEG-6000.After treatment for 7 days,the drought resistance of the transgenic tobacco was higher than that of the wild type(WT).WT tobacco wilted gradually and the leaves turned yellow under drought stress.Dry and dry,while the TP tobacco can better maintain the green leaves,the plant did not appear significant wilting phenomenon,TP tobacco has higher chlorophyll and soluble sugar content,photosynthesis is also stronger than the wild type.The activities of SOD,POD and CAT in TP tobacco were increased by 1.93,2.81,and 2.13 times compared to WT tobacco,and the MDA content was decreased by 2.88 times compared to WT tobacco,indicating that TP tobacco may increase protection enzymes in drought stress environment.4.Effect of HsPQ Gene Expression on Heat Tolerance and Cold Tolerance of Lotus lentifloraUnder 37? stress environment,the leaves of WT Lotus plants became yellow and dry,gradually shrivelled,and the damage was more serious than that of TP plants.The same transient osmotic treatment showed that TP had a higher chlorophyll content and soluble sugar content.The activities of the protective enzymes SOD,POD and CAT were 1.46,1.90 and 1.42 times higher than that of the WT lotus root,respectively,while the MDA content was reduced 1.38 times.In 4? stress environment,the leaves of WT Lotus began to appear curly,and the damage was more severe than that of TP plants.In the same environment,the TP of Lotus corniculatus treated with transient expression for 3 days had higher content of chlorophyll and soluble sugar,and its protective enzyme SOD.The activities of POD,POD and CAT were increased by 2.08,1.35 and 1.33 times compared with WT respectively,while the content of MDA was decreased by 1.33 times.Studies have shown that transgenic HsPQ tobacco grows better at 37? and 4? than WT.5.Effect of HsPQ Gene Expression on Drought Resistance and Salt Tolerance of Lotus japonicusWhen treated with 10% PEG-6000 drought stress for 7 days,the leaves of WT Lotus plants gradually wilted and yellowed,but the TP plants could maintain the green color well without obvious wilting.High chlorophyll content,soluble sugar content and strong photosynthetic performance showed that the activities of SOD,POD and CAT increased by 1.71,2.45 and 1.74 times compared with WT,respectively,while the MDA content decreased by 2.35 times.When treated with 150 mmol/L NaCl solution for 7 days under salt stress,it was observed that the leaves of WT Lotus began to appear yellow and a little drop,while TP could maintain its leaf green well;TP had higher chlorophyll and With soluble sugar content and strong photosynthetic capacity,SOD,POD,and CAT activities were increased by 1.75,2.18,and 1.43 times,respectively,compared to WT Lotus brasiliensis root,whereas MDA content was reduced by 2.22 times.Studies have shown that the HsPQ gene,Lotus corniculatum,grows better in the drought and salt stress environment than the WT Lotus plant,and the expression of this gene improves the stress tolerance of the recipient plants.6.The Relationship between HsPQ Gene Expression and Stress Resistance in Transgenic PlantsReal-time PCR analysis showed that the expression level of HsPQ exogenous genes was related to the stress tolerance of transgenic plants.The TP5 transgenic tobacco plants with stronger drought resistance had higher gene expression levels.In transgenic TP4 plants with better drought resistance and better salt-tolerant TP12 plants,HspQ gene expression was higher.