The Gene Expression Of NUP1、GEF And Relationship With Transport Efficiency Of Nicotine In Tobacco

Nicotine is an important factor which affect the quality of tobacco,It directly affect the availability of tobacco leaves.Conventional breeding methods was used to improve the nicotine quality in flue-cured tobacco varieties,but lack of effective screening reference for field selection.We want to find out useful screening reference to increase the selection efficiency.On the basis of the analysis,at the seedling stage,before the topping,and after the topping,using QRT-PCR to measure the expression of the Nt NUP1,Nt GEFgenes and Exploring the different contribution of Nicotine transporter specific protein gene(Nt NUP1),vesicular traffic regulator(Nt GEF)genes to nicotine accumulation at different growth period.It not only can provides reference for the early screening selection of high nicotine germplasms,but also can provide theoretical guidance for cultivating high-quality tobacco breeding nicotine varieties.In this study,13 agronomic traits and middle leaf’s nicotine content of 116 tobacco varieties were measured in 4 consecutive years.Various statistical analysis methods was used to explore the relationship and between nicotine and agronomic traits and and screen for agronomic traits related to nicotine content,On the basis of the nicotine content of 116 Flue-cured tobacco materials,Selecting 7 high and low nicotine flue-cured tobacco germplasms from 116 varieties to be plant by hydroponics,at the seedling stage,before the topping,and after the topping,using QRT-PCR to measure the expression of Nt NUP1,Nt GEF and other Nicotine metabolism genes(Nt PMT,Nt QPT,Nt MATE1/2,Nt JAT1/2).Exploring the different relationship between Nt NUP1,Nt GEF genes and nicotine content of leaves at different growth period.The main results as follows:(1)The variations in main agronomic traits of 116 tobacco varieties were abundant with the coefficients of variation ranging from 5.64%-16.96% and the variation coefficient of nicotine content was 24.3%.Simple correlation analysis showed that nicotine content was significantly positively correlated with the internodal distance,and negatively correlated with stem girth and leaf number.Factor analysis showed that leaf length-width ration factors,leaf number factors,leaf length factors,plant height factors were largely responsible for nicotine content.Regression models,which successfully forecasted the relationships between agronomic traits and nicotine content were established by Stepwise selection method analysis.Statistical test was performed and the results showed that the correlation coefficient was significant.Furtherly,second response surface analysis revealed the dynamical relationship of nicotine content between a leaf length and middle leaf length.When bottom leaf length is 63.7cm and middle leaf length is 58.7cm,the nicotine content of a tobacco plant is stable and has a minimal value.Nicotine content will increase when tobacco plants have a smaller leaf length-width ratio,bigger internodal distance,less leaf number,and higher plant height.(2)RT-PCR results showed that vesicular traffic regulator(Nt GEF)expressed strongly in leaf and root,But Nt NUP1 expressed strongly in root,weakly in leaf.What’s more,Nt MATE1/2 and Nt PMT related to nicotine metabolism were not expressed in leaf,the principal component analysis showed that NUP1 transporter is the major protein of nicotine transport in the root,but MATE1/2 transporter has highest nicotine transport efficiency,followed by JAT1 transporter.The vesicular traffic regulator(Nt GEF)and two nicotine synthesis genes(Nt PMT、Nt QPT)in root were the main factors affecting the nicotine content in leaves at the seedling stage.Besides,JAT1 transporter is the major protein of nicotine transport in the leaf at seedling stage,and the Nt QPT gene in leaves is also the main factor affecting nicotine content.the network diagram of nicotine and nicotine metabolic genes shows that Nt PMT gene in roots is the main factor that affects the nicotine content in leaves.The expression of Nt MATE1/2 in root promoted the expression of two nicotine synthetic genes(Nt PMT,Nt QPT)at seedling stages.(3)QRT-PCR results showed that Nt NUP1,Nt GEF and other Nicotine metabolism genes(Nt PMT,Nt QPT,Nt MATE1/2,Nt JAT1/2)were up-expressed after topping.Principal component analysis showed that NUP1 transporter has highest nicotine transport efficiency in root,followed by JAT1 transporter,MATE1/2 transporter has lowest nicotine transport efficiency at mature period.The vesicular traffic regulator(Nt GEF)in leaves can indirectly affected the accumulation of nicotine of middle leaves.In addition,The expression of Nt GEF in the root has the greatest effect on the two transporters of Nt MATE1/2 and Nt JAT1.Correlation analysis showed that JAT1 transporter has highest nicotine transport efficiency in leaves,what’s more,the expression of Nt NUP1 and Nt MATE1/2 were closely related to two major nicotinic biosynthetic genes(NtPMT and NtQPT).