| Salvia miltiorrhiza Bunge(S.miltiorrhiza Bunge),as one of the traditional Chinese medicines in China,is mainly used to treat cardiovascular and cerebrovascular diseases distributed in China and some Asian countries.The hydrophilic properties of S.miltiorrhiza of hydrophilic ingredients are more in line with the characteristics of traditional Chinese medicine decoction and are of great concern.The plant Psb D plays an important role in stabilizing the PS II complex on thylakoid membranes and affects plant photosynthesis.The photosynthesis products produced by plants through photosynthesis provide energy and precursor substances for secondary metabolites.Secondary metabolism is connected to primary metabolism from several main branch points.Therefore,the amount of photosynthesis products directly affects downstream secondary metabolism.In this study,the effect of overexpressing SmPsbD on photosynthesis of S.miltiorrhiza and its product synthesis was studied using tissue culture plants as the test material.We analyzed the causes of the changes in the amount of product synthesis,and further explored the effects of photosynthesis products on the biosynthesis of phenolic compounds in S.miltiorrhiza and their mechanisms.The main results obtained are as follows:1.We clarified the evolutionary relationship and tissue specificity of SmPsbD of S.miltiorrhiza.Phylogenetic tree analysis was performed on the amino acid sequence of SmPsbD of S.miltiorrhiza using MEGA7.0.The results showed that the target gene basically followed the evolutionary relationship from lower algae plants to higher dicotyledons.RT-q PCR was used to detect the transcription level expression of SmPsbD in the roots,stems,young leaves,mature leaves and flowers of S.miltiorrhiza at the flowering stage.It was found that the gene was not expressed in the roots,but was only expressed in above-ground tissues.The expression levels were: mature leaves > young leaves > stems > flowers.2.We successfully obtained SmPsbD overexpression transgenic plants.Recombinant plasmids of SmPsbD were constructed,and positive transgenic plants were obtained by using the plant genetic transformation system mediated by Agrobacterium tumefaciens GV3101.The corresponding genetic transformation efficiency of p CAMBIA1304 vector and overexpressing SmPsbD transgenic plants were 18.3% and 15.0%,respectively.RT-q PCR was used to analyze the relative expression of SmPsbD in the over-expressing transgenic plants.The results showed that Agrobacterium-mediated genetic transformation increased the transcription level of SmPsbD.3.We analyzed the changes in transcription levels of SmPsbD overexpression transgenic plants in S.miltiorrhiza.Comparing the transcriptomes of SmPsbD over-expression transgenic plants and wild-type S.miltiorrhiza found that a total of 11,354 differentially expressed genes(DEGs)(FC ? 2 and FDR ? 0.001)were generated,of which 5,084 DEGs were up-regulated and 6,270 DEGs were down-regulated.KEGG enrichment analysis of DEGs(P-value ? 0.05)found that DEGs are mainly concentrated in the following types of related metabolic pathways.The metabolic pathways related to photosynthesis,such as "photosynthesis","photosynthesisantenna protein" and "porphyrin and chlorophyll metabolism".The metabolic pathways related to signal transduction,such as "plant MAPK signaling pathway".The metabolic pathways related to photosynthesis products,such as "starch and sucrose metabolism".The metabolism related to secondary metabolism pathways,such as "phenylalanine metabolism","phenylpropane biosynthesis","tyrosine metabolism",and "flavonoid biosynthesis" and other metabolic pathways.Using Map Man software to visualize the metabolic pathways of DEGs,the results were consistent with the KEGG enrichment analysis of the transcriptome.The metabolic pathways related to photosynthesis and photosynthetic products were photosynthetic photosynthesis,sucrose-starch metabolism,amino acid metabolism,lipid metabolism and TCA cycle.Metabolic pathways related to secondary metabolism include flavonoid metabolism,terpenoid metabolism,and phenylalanine and phenol metabolism.The RT-q PCR technology was used to verify the gene expression related to light reaction in the transcriptome data.The results showed that the analysis results of RT-q PCR and RNA-seq had the same trend,indicating that the transcriptome data was reliable.4.We clarified that overexpression of SmPsbD of S.miltiorrhiza leads to differences in metabolites.Comparing the metabolome differences between SmPsbD overexpressing transgenic plants and wild-type S.miltiorrhiza(VIP ? 1 and FC ? 1.2 or FC ? 0.83),a total of 404 differential metabolites were screened out,of which 278 metabolites increased and 126 metabolites decreased.Using metabolanalyst3.0 software to perform KEGG enrichment analysis of differential metabolites,the results show that the metabolic pathways that are significantly enriched fall into two categories.Metabolic pathways related to primary metabolism include,biosynthesis of amino acid,pentose phosphate pathway,pantothenate acid and Co A biosynthesis and sugar biosynthesis.The metabolic pathways related to secondary metabolism include tyrosine metabolism,phenylpropane biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis," glucosinolate biosynthesis,plant secondary metabolism biosynthesis,plant hormone biosynthesis,biosynthesis of alkaloid derived from shikimate pathway and " ascorbate and aldarate metabolism and so on.5.We clarified the regulatory effect of SmPsbD on photosynthesis of S.miltiorrhiza and its products.Compared with wild type,the overexpressing transgenic plants of SmPsbD significantly increased the number of chloroplasts,chlorophyll content,photosynthesis parameters and chlorophyll fluorescence parameters,enhanced photosynthesis capacity,and increased photosynthesis product content.Combined with transcriptome DEGs,it was found that the up-regulation of the expression of related enzyme genes during chlorophyll biosynthesis may be the reason for the increase in chlorophyll content.The increase in the number of chloroplasts,the increase in chlorophyll content,the enhancement of plant photosynthetic capacity,and the up-regulation of key enzyme gene expression in primary metabolism-related pathways(such as the TCA cycle,sucrose-starch metabolism,and shikimic acid and chorismic acid metabolism pathways)may be the reason for the increased content of photosynthesis products.6.We clarified the regulation effect of SmPsbD of S.miltiorrhiza on phenolic compounds biosynthesis.Compared with the wild type,over-expressing of SmPsbD promoted the biosynthesis and accumulation of phenolic compounds such as salvianolic acid B,rosmarinic acid,total phenol and total flavonoids.The content of salvianolic acid B,rosmarinic acid,total phenol and total flavonoids in the over-expressing SmPsbD transgenic plants were 1.95?2.43 times,6.57?9.38 times,2.20?3.67 times and 1.87?3.39 times of the wild type,respectively.There are two main reasons for the increase in phenolic compounds biosynthesis.On the one hand,the increased biosynthetic accumulation of primary metabolites(shikimic acid,phenylalanine,tyrosine,etc.)in the shikimic acid and chorismic acid metabolism pathways provides sufficient substrate for secondary metabolism.On the other hand,overexpression of SmPsbD up-regulates the expression levels of key enzyme genes(PAL?4CL?RAS?CYP98A14?CHS?FLS?DFR and LAR)in the biosynthesis of phenolic compounds.This study revealed the effects of SmPsbD on the photosynthesis of S.miltiorrhiza and its products from physiological level,transcription level and metabolic level,as well as the positive regulation of photosynthesis products on the biosynthesis of phenol secondary metabolites.It provides a theoretical basis for improving the yield of S.miltiorrhiza by molecular means,and also lays a foundation for the metabolic regulation of active ingredients of S.miltiorrhiza. |
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