Establishment Of Transient Transformation System Of Sophora Alopecuroides L. And Foundation Analysis Of SaLDC Promoter

Establishing the genetic transformation system of medicinal plant is important to study their functional genes.6 factors of transformation were optimized,that is the concentration of agrobacterium tumefaciens,the infection time,the co-cultivation time of agrobacterium tumefaciens and S.alopecuroides callus,the preculture time of S.alopecuroides callus,the adding method of acetosyringone(AS)and the concentration of AS,respectively.The effect of different factors on transient transformation of agrobacterium study was to establish the optimal transient transformation system.Based on the coding region of lysine decarboxylase gene(SaLDC),the promoter sequence of the gene was cloned,and the SaLDC promoter was predicted by bioinformatics analysis.Deletion analysis of SaLDC promoter by transient expression of GUS in S.alopecuroides callus was coined out to determine the core region of the promoter and to study the regulation of different deletion promoters on downstream genes.Exploring expression pattern of SaLDC by genetic transformation of Arabidopsis thaliana was to provide evidence for cis-acting elements and its interactions relevant transcription factors.(1)A maximum transient transformation efficiency of 83.33%was achieved with 15d-precultured of S.alopecuroides callus,which was infected by A600=0.9 agrobacterium tumefaciens for 15 minutes and then co-cultivated for 48 hours with 200 μmol·L-1AS.(2)The promoter sequence(1260 bp)of upstream SaLDC was cloned from S.alopecuroides genomic DNA(gene bank accession number:KY038928).The result showed that it contained TATA-box,CAAT-box and others cis-acting elements,such as pathogen responsive element,drought and ABA responsive element,light responsive elementand,defense and stress responsiveness,MeJA responsive element,tissue-specific expression element and so on by bioinformatics analysis.It showed that SaLDC gene might influence the expression of lysine decarboxylase by response to biotic or abiotic induction.(3)The deletion fragment of SaLDC promoter with different lenghth(310、594、765、924 and 1260 bp)were ligated with the GUS reporter gene to form five plant expression vectors named P310、P594、P765、P924 and P1260,which were then transferred into S.alopecuroides callus.The GUS transient expression showed that all 5 different deletion fragments of SaLDC promoter could drive the GUS gene expression in S.alopecuroides callus.The SaLDC promoter we cloned had high promoter activity,however,there were significant different in the transient expression of GUS.The GUS transient expression rate of 310 bp promoter was the highest among the 5 promoter fragments,and it was decreased with the increase of the length of SaLDC promoter fragment.(4)Drought and light could induce the expression of GUS,therefore the SaLDC promoter was an inducible promoter.The predominant expression of GUS gene was in leaf and calyx driven by SaLDC promoter to indicate that it had the tissue-specific expression in transgenic Arabidopsis thaliana.GUS activity could be detected at different growth stages of seedling stage in transgenic SaLDC promoter Arabidopsis thaliana,however the expression level is different,and the expression level decreased with the growth of Arabidopsis,therefore the SaLDC promoter had the specificity of spatiotemporal expression.