Cloning Of Key Enzyme Genes In Monoterpene Biosynthesis And Studies On Monoterpenoid Synthesis Of Schizonepeta Tenuifolia Briq

SCHIZONEPETAE HERB A,an annual herbaceous plant in the Lamiaceae family,is the dry aerial part of Schizonepeta tenuifolia Briq.The monoterpenoids account for nearly 90%of its volatile oil,which largely determines the quality of the medicinal materials of S.tenuifolia.The quality of Chinese medicine directly affects the safety and effectiveness of clinical medication.Therefore,exploring the expression patterns and regulation mechanisms of key enzyme genes that affect the biosynthesis of monoterpenoids from the perspectives of molecular biology and genetic engineering has important theoretical value and practical significance for elucidating the biosynthesis mechanism of monoterpenoids and optimizing medicinal species of S.tenuifolia.This study first summarized the biosynthetic pathways of monoterpenoids in medicinal plants and related literature on key enzymes at home and abroad.At the same time,it summarized and analyzed the functions,regulatory mechanisms and effects of the exogenous elicitor MeJA on plants.Secondly,the StDXS1,StDXS2 and StLS genes were cloned by RT-PCR,and their bioinformatics analysis and time-space expression pattern analysis were carried out;the tissue culture and rapid propagation system of S.tenuifolia was established,and different concentrations of MeJA were used for different durations of treatment of explants.The change rule of glandular trichome phenotype and monoterpenoid accumulation of the plants after induction treatment were analyzed.Finally,the agrobacterium-mediated method was used to obtain the over-expressing StLS transgenic seedlings of S.tenuifolia.This study lays the foundation for clarifying the expression pattern and regulation mechanism of key enzyme genes in the biosynthesis of monoterpenoids in S.tenuifolia.The specific research contents and results are as follows:1.Cloning and bioinformatics analysis of StDXS and StLS genesIn this chapter,based on the early-stage S.tenuifolia.transcriptome data,RT-PCR was used to clone the first key enzymes StDXS1,StDXS2 genes in the terpene backbone biosynthesis MEP pathway and the first enzyme StLS gene in menthane monoterpene biosynthetic pathways,and used a variety of software for bioinformatics analysis.The results showed that the StDXSl and StDXS2 genes of S.tenuifolia.belong to the DeoxyxyluLose-5-phosphate synthase(IPR005477)family,which is mainly involved in the biosynthesis of terpenoids.This family is divided into 3 subfamilies,among which StDXSl and StDXS1 are clustered in DXS1 and DXS2 branches respectively.Through multiple sequence alignment and conservative motif analysis,it is found that the DXS genes of different subfamilies all contain the conserved domains "DRAG" or "TSAG",and the distribution of conservative motifs in different subfamilies is somewhat different.These different conservative motifs may determine to some extent DXS gene subfamily performs different biological functions.The StLS belongs to the Isoprenoid_Biosyn_C1 superfamily,which is mainly involved in the synthesis of isoprenoid compounds.The gene sequence contains terpene synthase-related domains and has the highly conserved monoterpene synthase sequences"RRX8W" and "DDX2D".The phylogenetic analysis showed that the StLS has a close evolutionary relationship with Agastache rugosa in the same family,and did not form a branch with the Mentha,indicating that the LS gene may have functional differences in different plants.The analysis of codon preference shows that the difference in codon preference between StLS gene and Arabidopsis is small,indicating that Arabidopsis is more suitable for subsequent heterologous expression studies of this gene.2.Comparative analysis of temporal and spatial expression patterns of StDXS and StLS genes and accumulation patterns of monoterpenoidsIn this chapter,we conducted a comparative analysis of the spatiotemporal expression patterns of the StDXS1,StDXS2 and StLS genes and the accumulation patterns of monoterpenoids.This part of the study is based on real-time fluorescent quantitative PCR(qRT-PCR),three commonly used candidate internal reference genes ACT,TUA and EF-1α were first cloned,and then EF-1α with better amplification efficiency and stability conditions was screened as the internal reference gene to analyze the spatiotemporal expression patterns of StDXS1,StDXS2 and StLS genes in different tissues(roots,stems,leaves,spikes)and different growth and development stages.At the same time,gas-mass spectrometry(GC-MS)technology was used to detect the types and contents of main monoterpenoids in different tissues and different growth stages of S.tenuifolia.Fifteen kinds of volatile oil components of leaves and 9 kinds of volatile oil components of PGTs were identified.Analysis of the temporal and spatial distribution of the content of the volatile oil components of S.tenuifolia can be obtained.With the growth and development of the plants,there are 4 main monoterpenoids.The content of(+)-limonene,(-)-isopulegone and(-)-pulegone in the ingredients showed a gradually decreasing trend,and the content of(+)-menthone showed a gradually increasing trend;In different tissues of the plant,the root contains very few monoterpenoids.The content of(+)-limonene,(-)-isopulegone and(-)-pulegone is distributed in the order of spike>leaf>stem>root,(+)-Mentone content distribution is leaf>spike>stem>root.With the growth and development of the plants,the relative expression levels of StDXSl and StDXS2 showed a trend of gradual decrease,while the expression levels of StDXS2 showed a slight increase trend at 118 d(the withering period of spikes).The relative expression level of StDXS1 in different tissues is spike>stem>leaf>root,and the relative expression level of StDXS2 in different tissues is root>spike>leaf>stem.The relative expression level of StLS gradually decreased with the growth and development of S.tenuifolia.The relative expression level in different tissues was spike>leaf>stem>root.The temporal and spatial expression pattern of StLS is very similar to the accumulation pattern of(+)-limonene,(-)-isopulegone and(-)-pulegone,which may be directly involved in the biosynthesis of some monoterpenoids.3.The effect of MeJA on the accumulation of PGTs and monoterpenoids in tissue cultured seedlings of S.tenuifolia.In this chapter,the establishment of a tissue culture rapid propagation system for S.tenuifolia for the first time,using different concentrations of MeJA to induce different time lengths of explants,and it was found that MeJA of medium and low concentrations may promote the growth and development of bush adventitious buds,the high concentration of MeJA affects the normal development of adventitious buds,resulting in a decrease in the proliferation rate.The density of PGTs in leaves under the treatment of 100～200 μmol/L MeJA increased significantly,especially the distribution of PGTs at abaxial of the leaves,and the diameter of PGTs increased.MeJA at a higher concentration of 300 μmol/L can significantly reduce the density of PGTs in leaves,reduce the distribution of PGTs at proximal of the leaves,and negatively regulate the growth and development of PGTs.GC-MS was used to detect the volatile components of the adventitious buds,and a total of 17 compounds were identified,of which the sum of 4 main monoterpenoids accounted for about 80-90%of the total volatile oil.After treatment with different concentrations of MeJA for different periods of time,MeJA was found positive regulation of(+)-limonene and(+)-menthol,and negative regulation of accumulation of(-)-isopulegone and(-)-pulegone.It may be that the(+)-limonene and(-)-pulegone components in the upstream of the biosynthetic pathway of menthane monoterpenes are more converted to the downstream(+)-menthone.4.Agrobacterium-mediated transgenic study of StLS in S.tenuifolia.In this chapter,the plant binary expression vector PBI121:35S:StLS:GFP:NOS containing the target gene d-Limonene synthase(StLS)and the reporter gene green fluorescent protein(GFP)was constructed.The S.tenuifolia were transformed with Agrobacterium LBA4404,and the resistant regeneration adventitious buds were obtained after 30 days of transformation through infection,co-cultivation,adventitious bud regeneration and other steps.After resistance screening and fluorescence irradiation screening,it was initially confirmed that they had been successfully transformed.The overexpression of StLS transgenic seedlings of S.tenuifolia was established.The results of this study lay the foundation for future research on the function of StLS gene in the process of menthane monoterpene biosynthesis,the improvement of species and the quality of medicinal materials of S.tenuifolia through genetic engineering.