The Effects Of Salt Stess On Seedling Development And Flavonoid Bisoysynthesis In Ginkgo Biloba

Ginkgo biloba is an important economic tree species in China.Leaves of ginkgo contain a variety of secondary metabolites such as flavonoids and terpene lactones,which are the raw materials for a variety of drugs.The planting on leaf gathering usually adopts seedlings in G.biloba.Salt stress is an important environmental factor affecting plant growth,and there are a large number of salted land distributed in China.This study took Ginkgo biloba seedlings as the research object,and adopted physiological,bioinformatics and molecular biology techniques to study the growth of Ginkgo biloba seedlings and the accumulation of secondary metabolites such as flavonoids under salt stress.The aim is to provide theoretical basis for the rational use of Ginkgo biloba cultivation in saline soil.The main results are as follows:（1）Under different concentrations of NaCl,the germination rate of Ginkgo biloba seeds decreased.Under low concentrations of salt stress（50 mmol/L and 100 mmol/L NaCl）,seeds could germinate,but the germination rate was much lower than that of control 40%.When NaCl concentration reached 200 mmol/L,seeds could not germinate.In the treatment of Ginkgo biloba seedlings,compared with the treatment of 200 mmol/L NaCl,50 mmol/L NaCl significantly promoted the survival rate of seedlings,and increased the growth of root,stem and leaf.（2）After 14 days seedlings were treated with NaCl at 50 and 100 mmol/L,the chlorophyll content and carotenoid content did not change significantly.However,under the treatment of 200 and 300 mmol/L,they decreased,especially,when the NaCl concentration was 300 mmol/L,total chlorophyll content decreased by 34%and carotenoid content decreased by 30%compared to the control.The soluble protein content in leaves increased with the increase of salt concentration.With the increase of salt concentration,the proline content of Ginkgo biloba leaves increased firstly and then decreased.When the NaCl concentration was 100 mmol/L,the proline content of leaves reached the lowest value.The contents of catalase（CAT）and peroxidase（POD）also increased first and then decreased with the increase of NaCl concentration.When the salt concentration was 50 mmol/L,the contents of CAT and POD reached the peak.In addition,the determination of flavonoid content in Ginkgo biloba leaves showed that 100 mmol/L NaCl treatment significantly increased the contents of total flavonoids and total flavonoid glycosides,especially the contents of quercetin and isorhamnetin.（3）The Illumina HiSeq 4000 sequencing platform was used to analyze the transcriptome of Ginkgo biloba leaves from the control group and the 100 mmol/L NaCl treated group,and a total of 1515 differentially expressed genes were identified.GO analysis was mainly concentrated in response to stess,defense response,fatty acid biosynthesis process and carbohydrate metabolism process.KEGG analysis was mainly concentrated in phenylpropanol biosynthesis,Zeatin biosynthesis and Ether lipid metabolism,as well as MAPK signaling pathway,Peroxisome,Fatty acid elongation,Plant hormone signal transduction and Flavonoid biosynthesis.（4）There were 4 differential genes related to signal recognition in salt signaling pathway,including receptor kinase gene and histidine kinase gene.There were 12 differential genes related to ion osmotic potential,including ABA signaling gene,CDPK/CIPK gene and MAPK signal transduction gene.In addition,transcription factors such as bZIP,WRKY,GRAS,AP2,MYB and NAC were also identified as involved in the response to salt treatment.The expression of transcription factors such as MYB（Gb₀6728）,bZIP（Gb₀2332）,WRKY（Gb 17623）,AP2（Gb₃7188）and NAC（Gb 37720）were significantly up-regulated by salt treatment.（5）A total of 8 differentially expressed genes were identified in the flavonoid biosynthesis pathway,among which the upstream and flavonol glycoside biosynthesis genes including PAL（Gb 10949）,FLS（Gb₀0285} Gb₄024）and OMT（Gb₂3214）showed up-regulated expression under salt treatment,while the key genes of anthocyanin biosynthesis.For example,DFR（Gb₂6459）and LAR（Gb₀8481 and Gb 40651）showed a down-regulated expression trend,qRT-PCR further confirmed that the expression of PAL and FLS genes significantly increased under salt treatment.（6）FLS is a key gene in the synthesis of flavonol glycosides in Ginkgo biloba.Further bioinformatics analysis with Arabidopsis FLS gene family evolutionary tree revealed that FLS（Gb₀0285）had a high homology with Arabidopsis AtFLS2 gene.So we named it as GbFLS2.The cis-acting elements analysis of the promoter revealed that GbFLS2 promoter had cis-acting elements related to defense and stress,as well as abscisic acid,auxin,gasmonic acid,anaerobic induction,meristem expression and other response elements.The GbFLS2 gene was amplified in Ginkgo biloba,and the amplified sequence was the genomic gene sequence of Ginkgo biloba with a length of 1086 bp.The overexpression vector of GbFLS2 gene was constructed and transferred into Ginkgo biloba callus.The results showed that the content of total flavonoids in Ginkgo biloba callus of GbFLS2 gene increased.In general,from the growth of Ginkgo biloba plant roots,stems and leaves,physiological indexes and flavonoid content,we screened out the more suitable growth condition of Ginkgo biloba seedlings under salt stress is 100 mmol/L salt concentration.Transcriptome data and qRT-PCR further demonstrated that Ginkgo biloba seedlings could improve salt tolerance by up-regulating transcription factors and key genes in salt stress signaling pathway under 100 mmol/L salt concentration.In addition,GbFLS2,a key structural gene in the flavonoid biosynthesis pathway was cloned.It was proved that GbFLS2 promoted the flavonoid biosynthesis by transforming Ginkgo biloba callus.