| Ginkgo(Ginkgo biloba L.)is a world famous relict plant,known as “living fossil”.Flavonoids and lactones are important secondary metabolites of ginkgo,which have anti-oxidant and anti-aging effects.At present,the research of flavonoid and lactone mainly focuses on the extraction of active substances and pharmacological components analysis,and the understanding of their biosynthetic pathway is insufficient.In this study,ginkgo leaves from10 original regions were used as research materials,and 3 individuals with high flavonoid content and 3 individuals with low flavonoid content were detected and selected as research objects.Transcriptome sequencing was used to compare and analyze the expression and regulation of flavonoid and lactone biosynthetic genes in two groups.To further elucidate the biosynthesis mechanism of flavonoid and terpene lactone in ginkgo leaves,the function of the important flavonoid and lactone genes were studied.The main results are as follows:1.Ginkgo leaves from 10 different original regions were used as materials to determine the total flavonoids,soluble sugar,protein contents and PAL(Phenylalanine ammonia lyase)activity,and the results showed that their average values were 9.13 mg/g,157.15 mg/g,0.71g/L and 10.96 U/g,respectively.Ginkgo flavonoid content was significantly positively correlated with soluble sugar content and PAL activity.Ginkgo leaves from Hanzhong had the highest flavonoid content,which should be paid more attention to in the selection of the source of excellent ginkgo leaves in the future.The variation coefficients of flavonoids in Duyun was the largest.Therefore,it is necessary to pay attention to the choice within the regions while choosing among the regions.2.According to the test results of individuals from different regions,three samples with high flavonoid content and three samples with low flavonoid content were selected to perform RNA-sequencing.A total of 533,952,528 clean reads were obtained from two groups of ginkgo leaves.After filtering and combining the sequences,37,625 unigenes were obtained,of which21,472(57.07%)were successfully annotated in five public databases.A total of 457 significantly differentially expressed genes(DEGs)were identified,of which 246(53.83%)DEGs were up-regulated in the high flavonoid content group.Among them,only two HMGS genes related to terpenoid biosynthesis were DEGs.A total of 53 unigenes were also identified to be related to the flavonoid biosynthesis pathway and 6 to the flavonol biosynthesis pathway.Moreover,based on ginkgo transcriptome data,22 SNP loci were screened for the establishment of the high-resolution melting(HRM)typing system and population verification.The results showed that the ginkgo HRM typing could achieve a better discrimination effect,which indicated the feasibility of SNP markers in population genetic diversity study of ginkgo and even trees.3.Based on the ginkgo transcriptome data and RACE techniques,we successfully cloned Gb F3'5'H1,Gb F3'H1,Gb FLS1 and Gb FLS2,four important enzyme genes with high expression levels in the flavonoid synthesis pathway.The expression levels of Gb F3'5'H1,Gb F3'H1 and Gb FLS2 were highest in leaves,and the expression levels of Gb FLS1 in stems and leaves were significantly higher than those in other tissues.The expression of leaves in different periods showed that the relative expression levels of Gb F3'5'H1,Gb FLS1 and Gb FLS2 were highest in April,while the expression levels of Gb F3'H1 were significantly higher in April and September than those in other periods.Non-targeted gas chromatography-mass spectrometry(GC-MS)analysis showed that Gb F3'5'H1 overexpression could increase the contents of 4',5-dihydroxy-7-glucosyloxyflavanone,epicatechin and gallocatechin in transgenic poplar,which were flavonoid-related.The overexpression of Gb F3'H1 significantly increased the content of flavonoids in transgenic plants,including epigallocatechin,gallocatechin and catechin.In addition,the leaves of transgenic poplar overexpressing Gb F3'H1 had a slight red pigmentation compared to non-transgenic plants.After overexpression of Gb FLS1,the contents of catechin,epicatechin,epigallocatechin and gallocatechin in the transgenic poplars were significantly lower than those of the control.And the expression levels of the three enzyme genes(DFR,ANS and LAR)were also significantly lower than the control,indicating that the synthesis of flavonoids has a negative regulatory effect.These results helped to better understand the molecular mechanisms of Gb F3'5'H1,Gb F3'H1 and Gb FLS1 in plant flavonoid biosynthesis and metabolism.4.Based on ginkgo transcriptome data,two differentially expressed HMGS genes in the pathways of sesquiterpene and triterpene synthesis were successfully cloned with the help of RACE technology,and their functions were investigated in vivo.The results of transient expression of Populus protoplasts demonstrated that the Gb HMGS1 protein was localized in the cytoplasm.The results of non-targeted GC-MS metabolomics revealed that there were 31 differential metabolites in the transgenic plants,which were significantly higher than those in non-transgenic plants.Specifically,there are three metabolites related to lactones,lanosterol(triterpenoid),dehydroabietic acid(diterpenoid)and plant alcohols(diterpenoid)contents were increased in the transgenic Populus plants.This suggests that the overexpression of HMGS1 may increase the synthesis of lactones. |
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