The Study On Vascular Specific Expression Of Quorum Quenching Enzyme AiiA

Vascular diseases,such as those caused by Ralstonia solanacearum and Fusarium oxysporum,can infect a wide range of plant species.These diseases are hard to control and commonly cause great economic losses.In recent years,plant genetic engineering,using various resistant genes,provides a new way for preventing vascular diseases.The N-acyl homoserine lactones encoded by the aii A gene could inactivate N-acyl homoserine lactones(AHLs)bacterial quorum sensing signals,thus blocking the expression of pathogenic genes,and finally quenching the bacterial virulence.This quorum quenching strategy provides a new prospective for the control of infectious bacterial diseases.The cauliflower mosaic virus CMV35 S promoter has been widely used in plant genetic engineering,which was shown to direct expression of target genes in most tissues and at various growth stages.Expression of target genes at a specific location and suitable time is an important aspect of research effort in transgenic engineering.In order to make better use of the aii A gene in prevention of vascular diseases,the promoter of xylogen protein At XYP1(Px)from Arabidopsis thaliana,is selected for evaluation of its ability in direction of vascular specific expression of target genes.The xylogen protein was shown previously to be expressed specifically in vascular tissues of A.thaliana,but the activity of its promoter and corresponding ability to direct tissue specific expression of target genes in other plants have not yet been reported.In view of its possible low strength,Px promoter was fused with 35 S promoter in tandem to prepare a construct 35S-Px to enhance the activity of Px.Firstly,six expression constructs were prepared i.e.,Z1(p BI121-Px-aii A),Z2(p BI121-35S-Px-aii A),Z3(p BI121-35S-aii A),Z4(PBI121-Px-GUS),Z5(PBI121-35S-Px-GUS),and 35S(PBI121-35S-GUS).A total of 13-85 transgenic tobacco lines from each construct were cultivated.The successful introduction of target sequences in the six constructs were verified by PCR using primers specific for aii A,uid A,Px,which showed that most of lines integrated the exogenous aii A,GUS,Px sequence into their genomes.Both promoter Px and 35S-Px regulated gene expression preferentially in vascular tissues.Histochemical GUS staining demonstrated that promoter Px,35S-Px was active in the xylem and phloem.Fusion of 35 S and Px can significantly enhance the strength of Px and increase its vascular specificity.The nine lines with higher expression of aii A gene among 20-42 Z1,Z2,and Z3 transplants were selected by Aii A enzyme activities,the aii A transcript expression and Aii A immunological analysis,which showed the aii A gene driven by the promoter Px,35S-Px and 35 S,can be transcribed and expressed in vascular tissues.Aii A enzyme activity in transline Z1,Z2 with promoter Px,35S-Px,were lower about 110,45 folds than those in transline Z3 with promoter 35 S.Compared with non-transgenic tobacco,the infection rate(4-5 grade)and the maceration length were obviously reduced in transline Z1,Z2 after inoculation with P.carotovorum E81,Transline Z1,Z2 showed a little or obvious stronger resistance for vascular infection than Z3 transplants,which indicated higher efficiency on concentrated expression of the Aii A enzyme in vascular tissue.The Aii A enzyme in transgenic-tobacco lines could obviously alleviate the bacterial damage in vascular tissues.In summary,the resistance gene aii A driven by the xylem-specific promoter Px could enhance the resistance to bacterial vascular infection and may save energy.It is feasible to use this approach to control AHLs-dependent vascular bacterial infections.