Preliminary Study On The Mechanism Of Ginseng WOXs Gene Regulating Ginsenoside Biosynthesis

Ginseng(Ginseng Radix et Rhizoma)is the dried root and rhizome of the Panax ginseng C.A.Mey.,which has the effects of nourishing vitality,replenishing the veins,removing spleen,benefiting the lungs,replenishing blood,and calming the nerves,is our precious Chinese medicinal materials.The previous research results of this research group show that the accumulation of ginseng "high quality",namely ginseng saponins,is of great significance to its "excellent shape",that is,the traits of roots.In order to further clarify the relationship between ginseng's external traits and intrinsic quality,this paper established a key transcription factor WOX(Wuschel-like homeobox)gene by screening the ginseng root type,and conducted a preliminary study on its mechanism involved in regulating ginsenoside biosynthesis,with a Lay the foundation for the research on the cause of ginseng's "excellent shape and high quality".In this paper,five WOXs gene over-expression and RNAi plant expression vectors were constructed,and PgWOX11 transgenic hairy roots were finally obtained through the transgenic hairy root system.Analysis of the gene expression of the transgenic material revealed that PgWOX11 gene has a positive regulatory effect on ERF1 B,ERF114b and PgIAA13 genes.Further,we analyzed the expression of the ERF family that may be regulated by PgWOX11,and found that the four ERF genes are likely to have a negative regulation effect on ginsenoside biosynthesis,of which ERF1 B positively regulated by PgWOX11.This study laid the foundation for elucidating the mechanism of ginseng WOXs gene regulation of ginsenoside biosynthesis,and provided a basis for exploring the relationship between ginseng's external traits and internal quality.The main research contents are as follows:1.Study on the response of ginseng WOXs gene to ginsenosides Adventitious roots of ginseng were treated with ginsenoside ginsenoside GS1 and ginsenoside ginsenoside GS2 and transcriptome analysis was performed.The results showed that(1)ginsenosides had a regulatory effect on the stem cell network of adventitious roots of ginseng,and the WOXs gene was a key gene of ginseng stem cells.On this basis,real-time fluorescent quantitative PCR was used to detect changes in the expression level of ginseng WOXs genes in different treatment groups.Five PgWOXs responded to ginsenoside treatment were obtained by screening.(2)Six ERF genes(ERF13,ERF113,ERF114 a,ERF114b,ERF110,and ERF1B)and one IAA13 gene consistent with the expression pattern of PgWOX11 were obtained by screening.2.Cloning and bioinformatics analysis of ginseng WOXs genes For the first time,five WOXs full-length genes were cloned from the total cDNA of ginseng roots,and were named as PgWOX4,PgWOX5,PgWOX11,PgWOX13 a,and PgWOX13 b.The proteins all contain the Homeobox domain.3.Construction of plant expression vector for ginseng WOXs gene and transgenic hairy roots Full-length cDNA and RNAi interference fragments were cloned from ginseng PgWOX4,PgWOX5,PgWOX11,PgWOX13 a,and PgWOX13 b genes.Overexpression and RNAi interference vectors were constructed using Gateway technology.Agrobacterium-mediated method was used to induce overexpression and RNAi to interfere with PgWOX11 transgenic hairy roots.PCR detection of GFP gene was positive.4.Study on the regulation of ginseng ERF by ginseng PgWOX11 Using transgenic hairy roots as controls,PgWOX11 overexpression and RNAi transgenic hairy roots were used to analyze the expressions of 6 genes that were consistent with the PgWOX11 expression pattern.The results showed that ERF1 B,ERF114b and PgIAA13 were highly expressed in PgWOX11 gene overexpression hairy roots,while ERF1 B,ERF114b and PgIAA13 were lowly expressed in PgWOX11 gene RNAi hairy roots.This indicates that ERF1 B,ERF114b and PgIAA13 are positively regulated by PgWOX11,and it is speculated that they may have a direct interaction relationship.Since ERF transcription factor is a key regulator of plant terpenoid metabolism,whether PgWOX11 affects ginsenoside synthesis through ERF transcription factor is a worth further exploration.5.Study on the regulation of ginsenoside biosynthesis by ginseng ERF gene Through heat map cluster analysis of ginseng transcriptome,six ERF genes that can participate in ginsenoside biosynthesis were screened from two parts of different parts and different growth years,namely ERF1 B,ERF003,ERF012,ERF026,ERF034,ERF118.Using methyl jasmonate to change the content of endogenous ginsenosides,the expression analysis of the six ERF genes screened showed that the expression levels of ERF003,ERF118,and ERF012 all significantly decreased,which was consistent with the trend of CYP716A53v2,and contrary to the trend of DDS,ERF1 B Contrary to CYP716A47 expression pattern.Combined with the correlation analysis,it can be inferred that ERF1 B is likely to be negatively regulated with CYP716A47;ERF003,ERF118,ERF012 is likely to be negatively regulated with DDS,and positively regulated with CYP716A53v2.Combining previous studies,ERF1B,ERF114b and PgIAA13 are positively regulated by PgWOX11.It can be speculated that PgWOX11 is likely to achieve the regulatory loop that regulates ginsenoside synthesis by regulating ERF1B.