Cloning And Expression Analysis Of Lysine Decarboxylase Gene In Sophora Alopecuroides L

As the first key enzyme gene involved in Matrine and Oxymatrin biosynthetic pathway, Lysine Decarboxylase gene played an important role in Sophora alopecuroides’ biochemical metabolism process.In this study, S alopecuroides.,an endemic Chinese crude drug in northwest China were collected as experimental materials.The coding sequence of LDC were cloned and the sequence polymorphism of different populations were analyzed,and correlation analysis was applied on the relationship between SNP sites and OMA content.The relationship between expression level of LDC and content of MA and OMA in young leaves and cotyledon was also studied.The promter fragment was also cloned in order to detect its function.All works endeavored to reveal the function of LDC,as well to lay a foundation for cultivation of high MA and OMA content varietis of S alopecuroides. by genetic engineering breeding.The results of the study were as below:(1) A full length of LDC CDS was isolated from S alopecuroides. using homologous cloning,named SaLDC(Acession Number:KM249871).It contains an open reading frame of 1368 bp encoding 455 amino acid residues and having a calculated molecular weight of 49.14 KD.Bioinformatics analyses indicated that the SaLDC CDS has 97% nucleotide sequence similarity with Echinosophora koreensis’ and Sophora flavescens’ which belongs to PLPDE_Ⅲ super family.Interestingly,the conserved amino acid residue(Phe340) in Quinolizidine Alkaloids-producing plants was spotted in the deduced amino acid sequence of SaLDC.Meanwhile,the cluster result showed that S alopecuroides. and other QAs-producing plants were clustered into one category.(2) 800 bp and 1500 bp fragment was isolated by Genome walking respectively.After sequencing and assembling, the SaLDC promoter of 1948 bp was cloned.Bioinformatics analyses indicated that the transcription initiation site was 45 bp upstream away from the start codon.Meanwhile,the TATA box and the CAAT box were located in the-27 bp and-81 bp.A lot of cis-acting element were found,including elements involved in light responsiveness, hormone responsiveness,tissue specific expression,defense and stress responsiveness.This many elements indicated that SaLDC exercises complicated biological functions in S alopecuroides.(3) The result of sequence polymorphism in 16 different populations showed that the variation mainly distributed in the 5’and 3’region.35 SNP sites were found in the CDS of SaLDC which had 13 synonymous mutation and 22 nonsynonymous mutation.The frequency of SNP is 1 SNP/39.08 bp.The Tajima’s D test and Ka/Ks ratio indicated the action of purifying selection in evolution.An association analysis of SNP site with OMA content were performed,6 SNP sites were found to be related to OMA content which could be used to develop CAPs primer.(4) Different mass fraction of PEG were used to stress the seeds and seedlings of S alopecuroides. The result showed that drought stress decreased the MA, OMA content and the expression level of SaLDC in cotyledon.However, the expression level of SaLDC increased under severe stress in cotyledon. The expression level of SaLDC and OMA content changed dynamically with time under drought stress in young leaves.The SaLDC expression and OMA content were inhibited under severe stress.While under mild and moderate stress,the OMA content in young leaves increased as SaLDC expression level increased,indicated that the expression of SaLDC regulates the MA and OMA content.