Metabolic Differences Of Taxus Cuspidata Based On GC-MS Non-targeted Metabonomics

Taxus cuspidata,also known as T.cuspidata and Pinus densiflora,belongs to Taxaceae.It is mainly distributed in Heilongjiang and Jilin provinces of China,and a small amount in Korea and Japan.T.cuspidata is a relict plant of the tertiary glacial period.The medicinal active substances of T.cuspidata,such as paclitaxel or harringtonine,have been widely studied,but the primary metabolites of T.cuspidata have been rarely studied.In September,wild T.cuspidata with the same diameter class and similar growth state were collected in Muling National Nature Reserve of Heilongjiang Province.At the same time,the wild T.cuspidata with the same diameter class and similar growth state were picked in Northeast Forestry University and cultivated.Based on GC-MS technology,the metabolism differences of bark tissue between wild and cultivated T cuspidata were analyzed,and the accumulation patterns of primary metabolites in different tissue parts(leaf and skin)of wild T.cuspidata were qualitatively analyzed.The main results are as follows(1)In this study,the primary metabolites of wild and cultivated T cuspidata were determined by GC-MS.Through the use of multivariate analysis,principal component analysis(PCA)and orthogonal partial least squares(OPLS-DA),45 kinds of significantly different primary metabolites were screened out,among which 4 kinds of amino acids including ornithine,citrulline,aspartic acid and o-acetylserine,and 10 kinds of amino acids were identified Organic acids include palmitic acid,stearic acid,4-hydroxybenzoic acid,ferulic acid,arachidonic acid,3-phenyllactic acid,galactose,xylitol,polyphenols and other compounds.Among them,23 metabolites with log2fc>0 were down regulated,including palmitic acid,4-hydroxybenzaldehyde,arachidonic acid,Dihydrocoumarin,citrulline,ornithine,2,4-dihydroxy benzaldehyde,4-diaminobutyric acid,cyclohexylformamide and so on,which indicated that the 23 significantly different primary metabolites were up-regulated in the other 22 differential metabolites log2fc<0,including Gentiobiose,d-arabinol,lysulose,epicatechin,ribose and so on.The results showed that the primary metabolites of wild T cuspidata and cultivated T.cuspidata were significantly different.(2)The obtained significant difference primary metabolites were input into KEGG metabolic pathway database to obtain the related metabolic pathways,and the metabolic enrichment analysis was carried out.A total of 22 metabolic pathways were detected.Through comprehensive analysis,we found that there were seven metabolic pathways with significant correlation,including sulfur metabolism,arginine and proline metabolism,pentose phosphate pathway,unsaturated fatty acid biosynthesis,galactose metabolism,alanine,aspartate and glutamine metabolism,and phenylpropanoid biosynthesis.The seven different metabolic pathways are generally divided into three types.The first type of amino acid and organic acid metabolism pathway includes sulfur metabolism,arginine and proline metabolism,unsaturated fatty acid biosynthesis,alanine,aspartate and glutamate metabolism.These metabolic pathways are related to protein and organic acid synthesis.The second type of pentose phosphate pathway and galactose metabolism are glucose metabolism.The third metabolic pathway is phenylpropanoid biosynthesis.(3)Most of the amino acids and organic acids in cultivated T.cuspidata are higher than those in wild T.cuspidata.Cultivated T cuspidata has more antioxidant substances and regulatory substances to alleviate stress,which can quickly alleviate and eliminate the damage and influence brought by stress such as drought and heavy metals,and better protect plant tissues and cells It has stronger resistance and adaptability to stress.The sugar accumulation of wild T.cuspidata was higher than that of cultivated T.cuspidata,which had better resistance to low temperature stress and could alleviate the harmful effects of stress environment in a more timely manner.(4)A total of 90 primary metabolites were identified from the skin and leaf tissues of wild T.cuspidata with different genders by GC-MS.Multivariate analysis was used to analyze the primary metabolites detected by GC-MS technology.Principal component analysis(PCA)and orthogonal partial least squares(OPLS-DA)digital models were used to analyze the skin and leaf tissues of wild T.cuspidata with different genders.After model test,the results showed that the samples of the same tissues of the same gender had obvious convergence,and the samples of different genders had the same characteristics There were significant differences in the tissue parts of each group,and the specificity was significant.(5)According to OPLS-DA digital model analysis and VIP value screening,53 primary metabolites were screened,including 3 sugars,19 organic acids,9 amino acids,10 alcohols and 12 other substances.Among them,30 kinds of primary metabolites were screened from skin tissue,including 12 kinds of organic acids,1 kind of sugar,4 kinds of amino acids,8 kinds of alcohols and 5 kinds of other compounds.26 different primary metabolites were screened out from leaf tissues,including 8 organic acids,3 sugars,6 amino acids,2 alcohols and 7 other compounds.The results of hierarchical cluster analysis showed that there were special primary metabolites accumulation in the skin and leaf tissues of wild T.cuspidata of different genders.In the skin tissues,there were 16 significantly different metabolites accumulation in male wild T.cuspidata,such as vanillic acid,cellobiose,galactitol,epicatechin,lactonic acid,l-tyramine Acid.The accumulation of the remaining 14 significantly different primary metabolites in female wild T.cuspidata was up-regulated.There were 12 different primary metabolites,such as β-alanine,phenylalanine,4-pyridoxine,6-hydroxynicotinic acid,d-sphingosine,3 amino acids,3 organic acids,1 alcohol and 5 other compounds,accumulated in the leaves of female wild T cuspidata.The remaining 14 metabolites accumulated significantly in male wild T.cuspidata,such as phenylalanine,isoleucine,serine,3,6-anhydro-d-galactose,cellobiose,fructotriose,4-aminobutyric acid,etc.amino acids and sugars accounted for the majority.