The Function Of Terpene Lactone In Stress And The Identification Of Key Genes In The Biosynthesis Of Terpenoids In Ginkgo Biloba

Ginkgo biloba L.is a unique relic plant in China.Ginkgo has significant economic value and is rich in secondary metabolites such as terpenoids.Terpenoids play an important role in human health and resistance to various stresses.Currently,research on the function of terpenoids mainly focuses on human health,with less attention paid to their function in stress resistance and key genes in synthesis pathways.In this study,1-5-year-old ginkgo trees were used as experimental materials.Through measuring the content of ginkgolides in leaves and assessing their antibacterial efficacy,we will further investigate the accumulation rules and function of terpenoids in stress resistance.The key genes of terpenoid synthesis were identified by RNA-Seq technology,and their functions were preliminarily analyzed.The main results are as follows:（1）The contents of ginkgolides in the leaves of ginkgo trees at different ages（2,3,and 5 years）were determined.Results indicated a negative correlation between total terpene lactones and tree age.Specifically,the levels of ginkgolides A and B were approximately 50%lower in the leaves of 2-year-old trees compared to those of 3-year-old and 5-year-old trees.Conversely,the content of bilobalide increased with tree age,and the content of bilobalide in the leaves of 2-year-old trees increased by 15.8%and 20.8%,respectively,compared with that of 3-year-old and 5-year-old trees.The difference is that the contents of ginkgolides and total terpene lactones in roots increase with tree age.Notably,the roots of 3year-old trees exhibited a significant increase in ginkgolides A,B,C and bilobalide,with respective increments of 32.6%,55.6%,109.3%,and 159.9%,compared to those of 2-yearold trees.The results showed that ginkgolides A and B in leaves decreased significantly with the increase in tree age,while ginkgolides C and ginkgolides in roots increased significantly with the increase in tree age.（2）The bacteriostatic experiment of ginkgolides on the mycelium growth of five pathogenic fungi showed that the crude extract of ginkgolides had the highest inhibition rate on Rhizotonia cerealis,with an inhibition rate of 18%in 0.02 mg/L medium and 50%in 0.04 mg/L medium.The inhibition rate of Verticillium dahliae was the lowest,and the inhibition rate reached 7.5%in 0.02 mg/L medium.In the medium with a concentration of 0.04 mg/L,the inhibition rate reached 21%.In addition,spraying 20 mg/L of G.biloba crude extract can improve the salt tolerance of G.biloba.It shows that the crude extract of ginkgolides plays an important role in resisting biotic and abiotic stresses.（3）Through transcriptome sequencing analysis of ginkgo leaves and roots with significantly different contents of terpenoids,8672 and 11007 differentially expressed genes were identified in the leaves and roots,respectively.GO functional enrichment analysis showed that the differentially expressed genes in the leaf and root comparison groups were mainly enriched in iron ion binding,DNA-binding transcription factor activity,transcriptional regulation activity,and antioxidant activity.KEGG pathway enrichment analysis showed that the differentially expressed genes in the leaf and root comparison groups were enriched in pathways such as Phenylpropanoid biosynthesis.Photosynthesis,Starch and sugar metabolism,Terpenoid backbone biosynthesis,Diterpenoid biosynthesis and other pathways.15 and 30 differentially expressed genes were enriched in the terpenoid synthesis pathway in the leaves and roots,respectively.Most of the differentially expressed genes in the MV A pathway were up-regulated in the roots,and the key structural gene Gb₂3677（GbHDR）in the MEP pathway also showed higher expression abundance in the roots,which was consistent with the trend of terpenoid content.GbHDR may be a key gene for terpenoid synthesis in ginkgo.In addition,combining transcriptome data with fluorescent quantitative results,Gb₃9714（GbGGPPS）was identified as highly expressed in the leaves and showed increasing expression abundance with increasing months,which was consistent with the accumulation pattern of ginkgolide A,suggesting that GbGGPPS may also play a key role in the synthesis of ginkgolide A in ginkgo.（4）GbHDR was cloned,an expression vector was constructed,and the results of subcellular localization showed that GbHDR was located in the cell membrane and nucleus.Transgenic Arabidopsis and tobacco were obtained by the inflorescence infection method and the leaf disc transformation method,respectively.Since ginkgolide is a unique component of Ginkgo biloba,the chlorophyll content of the downstream product of the MEP pathway in the overexpressed plants was detected,and the results showed that the chlorophyll content of the transgenic plants was significantly higher than that of the wild type.The Arabidopsis plants overexpressing GbHDR were compared with the wild type.The flowering period was earlier,the chlorophyll content was significantly higher than that of wild-type Arabidopsis plants,and the germination rate of Arabidopsis seeds overexpressing GbHDR on NaCl-containing medium was higher than that of wild-type plants,indicating that GbHDR can not only regulate the synthesis of terpenoids but also increase salt tolerance in plants.（5）GbGGPPS was cloned,an expression vector was constructed,and subcellular localization showed that GbGGPPS was localized in the cell membrane and nucleus.Transgenic Arabidopsis was obtained by the inflorescence infection method.Compared with wild-type plants,the transgenic plants had an earlier flowering period and significantly increased chlorophyll content.A statistical analysis performed on the salt stress tolerance of the overexpressed lines at the seedling stage and the germination rate of the transgenic lines.Significantly higher than the wild type,indicating that GbGGPPS also has the function of regulating terpenoid synthesis and enhancing salt tolerance.