Study On Chemical Composition Of Leaf Essential Oil And The Genes Related To The Biosynthesis Of Important Terpenoids In Cinnamomum Porrectum

Cinnamomum porrectum is an evergreen tree belongs to camphor(Lauraceae)and is well-known for its rich essential oil in leaves.The leaves of C.porrectum are rich in leaf essential oil and contain various terpenoids,which have been widely used in food,medical and daily-use chemical industries as raw materials.However,the chemical composition and variation of the essential oil and its biosynthetic pathways is not clear,which severely restricted the utilization of the main chemicals in essential oil,as well as the further study on genetic improvement.In this study,we collected the C.porrectum leaves in the main distribute of China,and the content,chemical compositions,distribution pattern,and annual change of leaf essential oil were studied.Based on this,we divided the chemotypes of C.porrectum and specified the main terpenoids types and characteristics of leaf essential oil.Furthermore,we analysed the biosynthesis pathway of three terpenoids including eucalyptol,camphor and linalool based on transcriptome sequencing data.Finally,we identified and cloned the key genes of biosynthesis pathway of the three terpenoids,analysed their expression patterns and verified the gene function.The main results are as follows:1.The content of leaf essential oil and its chemical compositions from 564 C.porrectum trees in 20 natural populations from 5 provinces were analysed.Leaf essential oil content among different populations and individuals were highly variable,has a range of 1.15-23.6mg/g and 0.2-41.3 mg/g,respectively.117 chemical compositions were detected in leaf essential oil of C.porrectum,including 51 monoterpenes,46 sesquiterpeneses and 20non-terpenoids.14 chemical types were divided in the main C.porrectum distribution areas.Among which,eucalyptol chemotype,camphor chemotype and linalool chemotype had the highest frequency on distribution.20 natural populations of C.porrectum were divided into 4categories by principal component analysis.The first category was Guangxi population with high essential oil content and wide distribution of eucalyptol chemotype.The second category was Yunnan population with wide distribution of camphor chemotype.The third category was the mixed population of Hunan population and Jiangxi population with wide distribution of elemol chemotype.The fourth category was the mixed population of Guangdong population and Jiangxi population featuring the diversified chemical types.This classification matched the geographical distances among different populations.2.Longitude,latitude,,altitude and annual average temperature of growing site are major environmental factors that influenced the variation of C.porrectum essential oil content and composition.The content of essential oil in C.porrectum had significantly negative correlation with longitude and latitude of their growing site.The content of linalool and ?-caryophyllene in essential oil had significantly positive correlation with annual average temperature of the growing site.The content of camphor showed significantly negative correlation with the longitude and latitude,but had significantly positive correlation with the altitude.The content of citral showed significantly positive correlation with the longitude and annual average temperature,but had significantly negative correlation with the latitude.3.The essential oil content in the C.porrectum leaf and the content of first principal component linalool in the leaf essential oil varied significantly across different months.The leaf essential oil content and the linalool content fell to the lowest level in March,but rose sharply in April.The leaf essential oil content was highest in May and maintained a relatively high level from April to August and a relatively low level in other months.However,the linalool content was quite stable throughout the whole year except in March.The leaf essential oil content had significantly positive correlation with the monthly average rainfall and the monthly average temperature.Both the high rainfall and monthly average temperature are beneficial to the synthesis and accumulation of leaf essential oil in C.porrectum.4.PacBio Sequel platform was used to carry out the third-generation full-length transcriptome sequencing for C.porrectum leaf tissue and 89 103 complete transcripts were obtained.Illumina HiSeq 2500 platform was used to conduct the RNA-Seq for eucalyptol chemotype,camphor chemotype and linalool chemotype leaf tissue of C.porrectum and high-quality expression profile data was obtained.From the terpene biosynthesis pathways,50,56 and 54 differentially expressed genes were screened from the eucalyptol chemotype vs.linalool chemotype group,the camphor chemotype vs.eucalyptol chemotype group,and the camphor chemotype vs.linalool chemotype group respectively.Meanwhile,21,22,18 differentially expressed genes were identified during the formation stages of terpenoids,including 7,9 and 5 genes related to monoterpene biosynthesis.Combined with the terpenoids synthesis pathway analysis and the qRT-PCR analysis of the differentially expressed genes,TPS-related genes CpTPS1,CpTPS3 and CpTPS4 in C.porrectum were selected to be the candidates for biosynthesis of camphor,eucalyptol and linalool.5.Based on the full-length transcriptome sequencing of C.porrectum,CpTPS1,CpTPS3 and CpTPS4 in C.porrectum were successfully cloned,and they coded 580,582 and 558 amino acids respectively.They all had the conserved DDXXD domains and(N/D)DXX(S/T)XXXE domains of TPS protein family.The phylogenetic analysis showed that CpTPS1 and CpTPS3 belonged to TPS-b subfamily and CpTPS4 belonged to TPS-g subfamily.Subcellular localization indicated that CpTPS1,CpTPS3 and CpTPS4 proteins were all located in chloroplast,and matched the synthesis location of the predicted monoterpenoids products,including camphor,eucalyptol and linalool.6.25 clones of 5 chemotypes were selected from the large C.porrectum group to verify the genetic function of CpTPS1,CpTPS3 and CpTPS4.The results showed that the expression levels for CpTPS1,CpTPS3 and CpTPS4 were positively correlated with the camphor,eucalyptol and linalool content.The gene expression and essential oil content analysis from the large group proved that CpTPS1,CpTPS3 and CpTPS4 were the key genes for biosynthesis of camphor,eucalyptol and linalool.CpTPS1,CpTPS3 and CpTPS4 over-expression vectors were constructed and successfully transferred into 84 K poplar(Populus Alba×P.glandulosa),and the over-expression could be detected.The results of GC-MS analysis of terpenoid compounds showed that,a small amount of linalool was detected in the leaves of five transgenic plants of CpTPS4,which proved that CpTPS4 was involved in the biosynthesis of linalool.