Preliminary Study On Candidate Genes Of Related Acyltransferases In The Aescin Synthesis Pathway Of Aesculus Chinensis Bge

Aesculus chinensis Bge,also known as Bimbisara tree,is a deciduous tree of heptaceae,which is widely distributed in China.Aescin as the main active ingredient of Chinese medicine sorrel,has extremely high medicinal value and can be used to treat cerebral hemorrhage,cerebral edema,respiratory diseases,etc.Aescin is a very important metabolite in the synthesis of triterpenes from Aesculus chinensis Bge.It is formed by multi-step glycosylation and acylation of the original aescin,and the later modification is directly related to the medicinal activity of the compound.In particular,acyltransferases are involved in acylation,catalyzing the transfer of acyl activated from the donor to the recipient.In this study,based on the analysis of the content of aescin(A,B)in different tissues of Aesculus chinensis Bge,transcriptome data and homology analogy,the acyltransferases involved in the aescin synthesis pathway were excavated.The expression of the candidate acyltransferase encoding factor was analyzed and the recombinant protein was purified,and to provide a theoretical basis for clarifying the synthesis mechanism of active ingredients of triterpenes.The results of this study are as follows:1.Quantitative analysis of aescin(A,B)in different tissues of Aesculus chinensis Bge by LC-MSAescin A and B are unevenly distributed in the tissues of horse chestnut,mainly distributed in flowers and seeds.The t test was performed on the content of aescin A in flowers and seeds,P <0.01,the difference between the two was very significant,and the content in seeds was significantly higher than that in flowers;meanwhile,the t test was performed on the content of aescin B in flowers and seeds,P <0.01,the difference between the two is also very significant,the content of seeds is also significantly higher than the flowers.Based on the differential accumulation of aescin in different tissues,it is speculated that changes in transcription levels play animportant role,so transcriptome differential analysis was used to screen high-expression acyltransferase genes.2.Discovery,analysis and verification of acyltransferase related genes in aescin synthesis pathwayBased on the analysis of transcriptome differences,this study used homology evolution analysis to screen for acyltransferase candidate genes.In the end,i successfully cloned and screened 11 acyltransferase-related genes,of which 3 were BAHD-ATs and 8 were SCPL-ATs.They were: AcBAHD 4,AcBAHD 25,AcBAHD26,AcBAHD 1,AcSCPL 7,AcSCPL 8,AcSCPL 9,AcSCPL 11,AcSCPL 15,AcSCPL16,AcSCPL 17.Further using qRT-PCR to detect their transcription in plants,the results showed that: AcBAHD 25 and AcBAHD 26 are the highest expressed in seeds,followed by flowers,and the expression in seeds is higher than flowers,the difference is very significant;while AcBAHD 4 The expression level in flowers is the highest,followed by seeds,and the expression level in flowers is higher than seeds,the difference is very significant.As for the acyl transferase genes of SCPL-ATs family,AcSCPL 7,AcSCPL 8,AcSCPL 15,AcSCPL 16,AcSCPL 17,the highest expression in seeds,of which AcSCPL7,AcSCPL8,AcSCPL16 are second only to seeds in flowers;AcSCPL1 has the highest expression in flowers,followed by seeds;AcSCPL9 and AcSCPL11 differ from the previous expression patterns in that they have the highest expression in leaves,followed by seeds.This is consistent with the results of flower and seed,which is the most accumulation site of aescin.It also verifies that these acyltransferase related genes are highly expressed in flower and seed,which provides another experimental basis for screening genes.In this thesis,prokaryotic and eukaryotic systems are used to explore the protein expression of candidate acyltransferases.The recombinant proteins(AcBAHD 4,AcBAHD 26)and yeast microsomes(AcSCPL 1,AcSCPL 8,AcSCPL 9,AcSCPL 11,AcSCPL 15,AcSCPL 16,AcSCPL 17)were obtained.