Improve Lupeol Biosynthesis In Arabidopsis Through Overexpression Of AtLUP1

Lupeol,a pentacyclic triterpenoid compound,is a special plant secondary metabolite which widely exists in diverse plant species such as fruits,vegetables and medicinal plants.It has been widely used in tumorprevention and treatment because of its pharmacological effects such as anti-oxidation,anti-malignant tumor proliferation,anti-inflammation,apoptosis induction and cholesterol reduction.However,the natural yield of lupeol is very low,and has to be directly extracted from plant tissues,which is destructive and highly depends on the plant resources.Chemical synthesis of such metabolites is limited by high costs,unfavorable by-products,and high toxicity.On the contrary,biotechnology with the advantages of production increment and cost reduction is going to be an important direction for the industrial production of bioactive substances.Lupeol synthase is a key enzyme for the biosynthesis of lupeol.Studies showed that it is difficult to obtain high conversion and yield rate of lupeol products by the strategy of bacteria or yeast engineering.It has been demonstrated that the LUP genes exist in Arabidopsis thaliana genome,however,the expression levels of LUP genes in Arabidopsis is quite low and no lupeol could be detected in the tissues of Arabidopsis.Based on this fact,we hypothesized that could the biosynthesis of lupeol be improved in plant cells by increasing the expression level of the lupeol synthase gene/s?In order to answer this question,in this study,we cloned an AtLUP1 gene from the Arabidopsis genome,constructed the over-expression system of AtLUP1,transformed the AtLUP1 gene into Arabdopsis to obtain AtLUPl over-expression plants,and detected lupeol content in the transgenic plants,to validatethe feasibility of this strategy.Finished works in this study are as follows:1.Cloning and identification of AtLUP1.The length of AtLUP1 fragment was 4302 bp,and its deduced CDS sequence was 2274 bp,which encodes a 757-amino acids protein product.Bioinformatics analysis showed that LUP was conserved in structure and function.2.Construction of overexpression system of AtLUP1 and genetic transformation.The AtLUP1 fragment and pBI121 vector were ligated to construct a plant expression vector which was used to transform Arabidopsis.Eighteen transgenic lines were obtained through screening and identification.No significant difference in physiology and morphology was not found among transgenic and wild-type lines,indicating that AtLUPl is not necessary for Arabidopsis,and the excessive expression would not affect the normal growth and development of Arabidopsis.3.Detection of lupeol in Arabidopsis by GC(gas chromatography).The content of lupeol in transgenic lines was analyzed by GC.The results showed that the content of lupeol in wild-type was very low(0.0033 mg/g),and the content of lupeol in transgenic lines are generally higher than that in the wild type.There are three strains with the lupeol content higher than 0.1mg/g;five strains between 0.05 and 0.1 mg/g;and ten strains lower than 0.05 mg/g.Among the transgenic lines,line No.48 had the highest lupeol content of 0.9155 mg/g,which is 277.4-fold of that in wild type;in No.49 and No.68,the lupenol content was 43.9 and 43.79-fold of the wild type;the lower lupenol content was the No.14,which was only 1.71-fold of the wild type.This study demonstrates that overexpression of AtLUPl could improve the biosynthesis of lupeol in plant cell,thus provides a potential direction for the industrial production of lupeol.