System Analysis And Functional Study Of NAC Transcription Factor In Panax Ginseng

Ginseng(Panax ginseng C.A.Mey),a valuable traditional Chinese medicine,is a perennial herb and belongs to the Araliaceae family.Ginsenosides is the main bioactive component in ginseng.Intensive studies have been conducted to reveal the molecular mechanism of ginsenoside biosynthesis during the past decades.As one of the largest and plant-specific transcription factors,NAC transcription factor gene family plays the vital role in process of growth,development,and response to stress.The NAC family has been well characterized in the model plants.However,little is known about the NAC family in ginseng.Therefore,identification,characterization and utilization of this transcription factor family in ginsenoside biosynthesisin ginseng are of significance.In the present study,based on the comprehensive analysis of NAC gene family in ginseng,ginsenoside biosynthesis-related NAC genes were seek out as candidate gene,followed genetic transformation using Agrobacterium rhizogenes mediated method,and analyzed the content of saponins in the conversion materials.Hoping the comprehensive data generated here can provide a reference for elucidating functions of NAC transcription factors family in ginseng.1.A total of 251 Pg NAC Unigenes were identified from the 248,993 Unigenes database of Jilin ginseng.Phylogenetic analysis revealed that the Pg NACs were clustered into 18 subfamilies and were highly conservative.2.The GO annotation results indicate that at the Level 2,Pg NACs are annotated into eight functional subcategories: single biological process,cellular process,metabolic process, binding,catalytic activity,cellular part,organelle,and cell.Enrichment analysis showed that Pg NACs were significantly enriched in cellular processes,catalytic activity,binding,and cellular parts.Analysis of expression patterns showed that the expression of Pg NACs has substantially diversified across different tissues,developmental stages and genotypes,showing up-regulation or down-regulation expression;network analysis showed that Pg NACs are closely linked and interact participate in the metabolic process of plants.3.5 stress-related Pg NAC genes(Pg NAC05-2,Pg NAC41-2,Pg NAC48,Pg NAC56-1,Pg NAC59) were selected,the ginseng hairy roots were cold-treated,and the expression pattern of genes was studied under cold stress by q RT-PCR,preliminary verification of its function in cold stress of ginseng.4.The correlation analysis between Pg NACs and ginsenoside content was performed,and the results showed that 94 Pg NACs genes were significantly correlated with the ginsenoside content;the correlation analysis between Pg NACs and the expression of key enzyme genes for ginsenoside synthesis showed that 122 Pg NACs genes were correlated with the expression of key enzyme genes.At the same time,there were 60 Pg NACs significantly related to ginsenoside content and expression of key enzyme genes,of which 24 Pg NACs contained complete ORFs and conserved domains.Under different P-value conditions(P value =5.00E-02 — P value = 1.00E-08),the network analysis of the 24 Pg NACs and the key enzyme genes for ginsenoside synthesis showed that Pg NAC41-2 is always closely related to the key enzyme genes,so Pg NAC41-2 was selected for further functional verification.5.Pg NAC41-2 gene was cloned and the overexpression vector was constructed.Agrobacterium rhizogenes mediated method was applied to performed the genetic transformation using leaf stalk as explants.The positive hairy root was obtained after PCR analysis.At last,positive hairy roots was get.6.Ginsenoside was extracted and purified from positive hairy roots,the content of monomer ginsenosides in positive hairy roots was determined with HPLC.The results showed that Pg NAC41-2 gene promote the biosynthesis of saponins.