Expression Characteristics And Functional Identification Of CYP450 Genes From Betula Platyphylla Suk.in Triterpenoid Synthesis

The bark of Betula platyphylla Suk.（Birch）contains betulin,betulinic acid,and oleanolic acid and other triterpenoids.Triterpenoids have a wide range of pharmacological activities,such as anti-virus,anti-tumor,anti-diabetic,anti-hyperlipidemic and anti-HIV.However,the natural sources of triterpenoids are limited,and they are easily affected by factors such as environment,tree age,and tissue specificity.The traditional method of obtaining birch triterpenes is not conducive to the sustainable utilization of natural products.Cytochrome P450monooxygenase（CYP450）is one of the key enzymes in the terpenoid synthesis pathway,and plays an important role in the synthesis and diversification of terpenes and saponins.CYP450 also plays a huge role in plant growth and development and in response to environmental stress.In this study,birch was used was the test material,9 birch Bp CYP450genes were cloned from the transcriptome database and bioinformatic analysis was performed.The expression patterns of 9 birch Bp CYP450 genes in different abiotic stress treatments and different tissues were detected.And 9 Bp CYP450 expression vectors of plant and yeast were constructed to detect the content changes of triterpenes and its components in recombinant yeast,tobacco and birch cells.This study is expected to further excavate and identify novel genes with high-efficiency catalysis of high value-added triterpenoids such as betulinic acid,oleanolic acid and their derivatives.The results of this study are as follows:1.9 Bp CYP450 genes with a length of 1509～1925 bp using the birch seedling c DNA as a template were cloned in this study.They are all new genes and are tentatively named:Bp CYP59,Bp CYP118,Bp CYP122,Bp CYP162,Bp CYP176,Bp CYP187,Bp CYP1,Bp CYP4,Bp CYP5.The longest ORF lengths of the cloned 9 Bp CYP450 genes were distributed in1431～1575 bp,encoding 486～524 amino acids,and all 9 Bp CYP450 genes had the conserved CYP450 domain.The Bp CYP59,Bp CYP118,Bp CYP122,Bp CYP1,Bp CYP5 proteins had high homology with the Morella rubra CYP71A25,CYP90A1,CYP76C4,CYP82D47 and CYP78A3 proteins,ranging from 73.85%to 91.56%.2.The tissue specificity of 9 Bp CYP450 genes and 5 key enzyme genes of triterpenoid synthesis were analyzed by real-time quantitative PCR.The results showed that Bp CYP59,Bp CYP176,Bp CYP1,and Bp CYP4 had similar tissue expression specificities,and the relative expression levels of the genes were stem>root>leaf,and the relative expression levels of the other five Bp CYP450 genes were stem>leaf>root.In order to explore the response of 9Bp CYP450 genes to hormones and mechanical damage,birch tissue culture seedlings were treated with 100μmol/L Me JA,50 mg/L SA,100 mg/L GA₃,10μmol/L ABA and mechanical damage.These results show that the 9 Bp CYP450 genes have different response patterns to hormonal and mechanical injury.The promoter sequences of 9 Bp CYP450 genes were analyzed.It is further demonstrated that the obtained nine genes can be induced by various hormones and may play important functions in resisting and adapting to abiotic stress in birch.3.In this study,9 Bp CYP450 genes were overexpressed in Saccharomyces cerevisiae Jy602-Bp W,and nine recombinant yeast strains Jy602-Bp W-CYP450s were obtained.The recombinant yeast strain Jy602-Bp W-Bp CYP1 was detected to have the function of synthesizing betulin,and the content reached 5.24 mg/L.The content of total triterpenoids in recombinant yeast increased significantly after transferring into Bp CYP118 and Bp CYP176,which were 4.19 times and 3.70 times higher than that of control Jy602-Bp W,respectively.In conclusion,Bp CYP118,Bp CYP176 and Bp CYP1 are play a more important role in the synthesis of triterpenoids in Saccharomyces cerevisiae.Nine Bp CYP450 genes showed different stress resistance functions in yeast.4.In order to verify the catalytic function of 9 Bp CYP450 genes on plant triterpene synthesis,transient overexpression was performed in tobacco and birch cells.In tobacco,after infection with Bp W+Bp CYP187 and Bp Y+Bp CYP118,the content of betulinic acid was 1.38mg/g（Dry weight,DW）and 1.52 mg/g（DW）,respectively.1.30 times and 1.43 times that of wild-type tobacco.The overexpression of Bp W+Bp CYP4 gene produced the highest oleanolic acid content,which was 4.13 mg/g（DW）,while the control tobacco did not produce oleanolic acid.It was clear that among the 9 genes,Bp CYP118 and Bp CYP187 could oxidize lupinol to produce betulinic acid.Bp CYP59,Bp CYP118,Bp CYP122,Bp CYP162,Bp CYP1,Bp CYP4genes have the function of oxidizingβ-amyrin to produce oleanolic acid.The Bp CYP162 gene can produce oleanolic acid and catalyze the production of betulin from lupinol;the Bp CYP118gene can produce oleanolic acid and catalyze the production of betulinic acid from lupinol.Further transient infection of birch cells showed that Bp CYP118,Bp CYP122,Bp CYP176,Bp CYP187 and Bp CYP1 genes could oxidizeβ-amyrin to obtain oleanolic acid.At the same time,when Bp W was co-expressed with Bp CYP118,Bp CYP122,and Bp CYP187,the oleanolic acid content was significantly higher than that of the wild type,reaching 0.68 mg/g,0.62 mg/g,and 0.71 mg/g（1.62 times,1.48 times,and 1.69 times that of uninfected birch cells）.The Bp CYP187 gene that transiently infected both tobacco and birch cells and produced higher content of triterpenoids was selected to stably transform tobacco,and the transgenic plants were obtained by screening and identification.The content of triterpenoids was detected by high performance liquid chromatography（HPLC）on transgenic tobacco tissue culture seedlings and wild type tissue culture seedlings.Oleanolic acid and betulin were not detected in wild type tobacco（culture seedlings）,and contents of betulin and oleanolic acid were 0.056mg/g（DW）and 0.015 mg/g（DW）in Bp CYP187 transgenic tobacco,respectively.It further indicated that Bp CYP187 gene has the function of promoting the synthesis of triterpenoids,and can catalyze the synthesis of oleanolic acid and betulin.In conclusion,Bp CYP118 and Bp CYP187 play an important role in the synthesis of birch triterpenoids.The results of this study provide important new genes resources for the biotechnological synthesis of high value-added and highly active triterpenes and saponins.