Optimization Of Lectin Accumulation System In Tobacco

Nicotiana tabacum lectin（Nictaba）is a plant lectin induced by jasmonic acid（JA）or methyl jasmonate（JAME）in tobacoo.Nictaba plays an important role in response to tobacco stress,is a natural protein that can be used to control pests and pathogens.The conditions of inducing Nictaba accumulation in tobacco leaves in vitro and the isolation and extraction of Nictaba have been reported,but few studies on Nictaba accumulation in tobacco varieties,growth period,spraying concentration of JAME and treatment time have been reported.In this study,q PCR was used to detect Nictaba gene expression in order to establish a system to optimize Nictaba accumulation cultivoir,growth period and induction conditions,and to provide reference for the development and utilization of Nictaba.The main findings of the study are as follows:（1）A method for separating and extracting lectin from tobacco was developed.In this experiment,tobacco cultivoir“Yanyan 97”was used as experimental object,two separation strategies were developed to purify Nictaba,the high efficiency of the method was discussed by yield,purity and resin performance-price ratio.The results showed that the active protein of tobacco leaf protein after affinity chromatography with thyroglobulin-Sephadex 4B resin was a single protein with an apparent molecular weight of about 58 KDa.The average recovery of Nictaba extract was 19.05%,the average yield was 339.68?g·g^-1FW.The average recovery and the average yield of chitin affinity chromatography were 22.29%and 365.04?g·g^-1FW,respectively.The protein obtained by the two methods had the same agglutination activity and molecular weight,but the chitin affinity chromatography was selected for the follow-up test.（2）The system of Nictaba accumulation was optimized based on q PCR.Taking tobacco cultivoir“Yanyan 97”as experimental object,the effects of growth stage,JAME concentration and treatment time on Nictaba accumulation were studied based on q PCR technique.Using response surface analysis and regression analysis of three factors（concentration,treatment time and growth stage）to establish mathematical models,the optimal system of Nictaba gene expression was optimized as follows:JAME induction concentration was 59.02?mol·L^-1,treatment time was 6.35 h,growth stage was 13.05 leaf stage.In theory,the expression of Nictaba gene was 20.39 times higher than that of Ntubc2 gene.Under these conditions,parallel experiments were repeated to verify the predicted value of the model,and the relative expression of Nictaba gene was 19.84 times,which was basically in agreement with the predicted value.（3）The protein content of Nictaba was determined to verify the optimized system for inducing Nictaba accumulation.Nictaba was induced and isolated by JAME at the key node of“Yanyan 97”growth stage.The results showed that Nictaba content was the highest at the 12-leaf elongation stage,reaching 364.97?g·g^-1FW,the results showed that there was a significant positive correlation between the gene expression level and the optimal induction of Nictaba accumulation system,which also shows that the optimized Nictaba cumulative system is accurate.In addition,the gene expression and protein content of Nictaba induced by JAME in six tobacco germplasm materials were detected.The results showed that the content of Nictaba in“Qinggeng”was the highest,reaching 415.87?g·g^-1FW,“Yanyan 97”and“MC994”were364.97?g·g^-1FW and 296.33?g·g^-1FW,respectively.Moreover the expression level of Nictaba gene was highly consistent with the content of Nictaba protein.It is concluded that“Qinggeng”,“Yanyan 97”and“MC994”are high expression cultivoir of tobacco lectin.