Screening Of Cuticular Wax Synthesis Gene Regulated By MIXTA1 Of Eustoma Grandiflorum

The Eustoma grandiflorum is native to the central and southern regions of the United States.Its flowers with a long lifespan are rich and diverse,and the stems are light and unrestrained.They have high ornamental and economic value.The surface of Eustoma grandiflorum leaves has a layer of cuticular wax,which acts as a hydrophobic protective barrier that contributes to the growth and adaption ability of Eustoma grandiflorum.The main components of cuticular wax are wax esters,aldehydes,ketones,alkanes,primary alcohols,secondary alcohols,and other substances.Research has found that the biosynthetic pathway of cuticular wax is regulated by many different genes,and these genes related to cuticular wax biosynthesis are regulated by different types of transcription factors.The study found that the overexpression of the EgMIXTA1 gene can increase the cuticular wax on the leaves of the Eustoma grandiflorum.To further verify that the EgMIXTA1 gene is involved in the biosynthesis of cuticular wax of Eustoma grandiflorum,we conducted experiments to screen out the candidate genes regulated by EgMIXTA1 from RNA-seq of the transgenic plants with EgMIXTAl overexpression and wild-type plants of Eustoma grandiflorum.To further verify the RNA-seq screening results,we obtained the information of candidate gene sequence from the Eustoma grandiflorum transcriptome and genome database and determined the gene name by using NCBI-BLAST and conducted the expression level analysis of candidate gene and tissue.Through the analysis of the candidate gene promoter sequence,a 2000 bp promoter sequence containing important functional elements and MYB transcription factor binding sites was selected for promoter cloning.Used chromatin immunoprecipitation(ChIP)and real-time quantitative PCR methods to initially verify the hypothesis,and then further verified by the dual-luciferase reporter system.Finally,the gene regulated by the EgMIXTA1 transcription factor was confirmed,and the gene was heterologous expressed in Arabidopsis to verify the gene function.The results of this research are as follows:(1)Use bioinformatics methods to determine the candidate gene names and sequence information obtained from the preliminary RNA-seq cluster analysis,consult the relevant literature to understand gene functions,and initially screen out nine candidate genes that may be regulated by EgMIXTA1 transcription factors:EgCER3,EgCER6,EgKCS3,EgKCR1,EgGAUT1,EgXTH1,EgSULTR2,EgPTAC16,EgABCG12.(2)Real-time quantitative PCR technology was used to verify the RNA-seq screening results.The candidate downstream genes EgCER3,EgCER6,EgKCS3,EgKCR1,EgGAUT1,EgXTH1,EgSULTR2,EgPTAC16 were all highly expressed in EgMIXTA1 overexpression plants,and the EgABCG12 gene expression level was consistent with that of the wild type which can be used as a control in subsequent experiments.(3)The tissue specificity of nine candidate genes was analyzed using real-time quantitative PCR technology.The candidate genes are all highly expressed in the leaves of Eustoma grandiflorum,and low or not expressed in the roots.The result is in line with that cuticular wax is the protection of the above-ground parts of plants and nine candidate genes are related to cuticular wax synthesis.(4)To clone the nine candidate genes with 2000 bp promoter containing important functional elements and MYB transcription factor binding sites in its sequence,we used the sequence information of nine candidate genes in the Eustoma grandiflorum genome database,obtained the important functional elements in the promoter sequences of the nine candidate genes and the binding sites of MYB transcription factors using bio informatics methods.(5)The 35S:EgMIXTAl-3×FLAG overexpression carrier was constructed,and ChIP-qPCR was performed using the prairie gentian protoplasts that transiently expressed the 35S:EgMIXTA1-3×FLAG overexpression carrier to verify that the downstream target genes regulated by the EgMIXTA1 transcription factor were EgCER3 and EgKCS3,EgKCR1,EgXTH1,EgSULTR2.(6)Construct a dual-luciferase reporter carrier with nine candidate gene promoters,and successfully construct 35S:EgMIXTA1-YFP expression carrier simultaneously.By transiently transforming 35S:EgMIXTA1-YFP expression carrier and dual-luciferase reporter carrier of candidate genes in Eustoma grandiflorum to further confirm that the downstream target gene regulated by the EgMIXTA1 transcription factor is EgCER3.(7)EgCER3 was cloned and successfully constructed 35S:EgCER3-3×MYC overexpression carrier;EgCER3 was heterologous expressed in Arabidopsis,and EgCER3 overexpression transgenic Arabidopsis seedlings were successfully obtained.