Characterization Of The Promoter Of The Broad Spectrum Powdery Mildew Resistance Gene NLR1-V In Haynaldia Villosa

Wheat powdery mildew is a fungal disease caused by Blumeria graminis f.sp.tritici.It is one of the most destructive wheat diseases in the world,which seriously affects the yield and quality of wheat.Up to now,12 powdery mildew resistance genes have been cloned,among which Pm21 is a broad-spectrum powdery mildew resistance gene derived from the short arm of 6V chromosome of wild species Haynaldia villosa（2n=2x=14,VV）.Pm21 shows high level of resistance or complete immune to almost all of the isolates of Bgt.Pm21 has become one of the most effective resistance genes introduced from wild species into common wheat,and has been widely used as an important resistance source in wheat breeding and production.At present,the powdery mildew resistance gene NLR1-V at Pm21locus has been finely mapped and cloned,which provides a prerequisite for the study of its broad-spectrum resistance mechanism.Promoter plays critical roles in regulating gene expression at the transcriptional level,such as the transcription starting time,expression mode and expression site.The gene promoter region generally contains cis-acting elements that regulate the specific expression of genes.Thereful,study on the composition and function of these expression regulatory elements in the promoter region is helpful to understand the regulation mechanism of gene expression at the transcriptional level.At present,the resistance mechanism of NLR1-V gene is poorly understood.In order to explore the regulation mechanism of NLR1-V gene at the gene expression level,we cloned the promoters of NLR1-V gene,determine the key regions with pathogen-induced activation activity.Meanwhile,the promoters of three orghologous gene of NLR1-V in common wheat were cloned,and their structural characteristics and functional differences were compared and analyzed to study the evolution process of alleles of NLR1-V in different genomes.At the same time,the key cis-elements of NLR1-V promoter that play a role in response to Bgt were explored,and the regulation mechanism of NLR1-V expression was preliminarily clarified,which provided an important theoretical basis for the analysis of broad-spectrum resistance of NLR1-V,and provided ideas for using NLR1-V in breeding.The main research results are as follows:1.Cloning and detecting the Bgt-responsive activity of the upstream sequence of NLR1-VBased on the assembled Sc18Q1Z＿893,the primers were designed to clone the sequence upstream ATG of NLR1-V,then the 2557bp sequence were recomninated into PWMB002 to construct the vector PWMB002:NLR1-Vpro:GUS,in which the GUS gene was drived by NLR1-Vpro.The activity of NLR1-Vpro was detected by transient expression technique mediated by gene gun bombardment.The results showed that the 2557bp sequence could significantly respond to the inoculation of Bgt.At the same time,we compared the transcripts of NLR1-V,NLR2-V and Sc18Q1Z＿893,then we defined the5’UTR region of NLR1-V gene from ATG to the upstream at 335bp,and the promoter region of NLR1-V gene from 335bp to 1067bp.Sequence analysis revealed a variety of tissue-specific expression elements,hormone-responsive elements and pathogen-induced elements.2.Screening the promoter region of NLR1-V gene in response to powdery mildewBased on the above research results,a series of promoter truncated plasmids w ere constructed by the 5’-end deletion method,including PWMB002:NLR1-Vpro_{1044-A TG}:GUS,PWMB002:NLR1-Vpro_889-ATG:GUS,PWMB002:NLR1-Vpro_668-ATG:GUS,PWMB002:NLR1-Vpro_474-ATG:GUS,PWMB002:NLR1-Vpro_316-ATG:GUS.The activity of all frag ments were detected by transient expression technique,and the results showed that a large numbers of GUS-expressing cells could be observed in the Bgt inoculated lea ves transformed with NLR1-Vpro_1044-ATG,NLR1-Vpro_889-ATGand NLR1-Vpro_668-ATG,whil e a little GUS-expressing cells could be observed in the non-inoculated leaves.In th e leaves transformed with NLR1-Vpro_474-ATGa small number of GUS expression cells could be observed in the Bgt inoculated and uninoculated samples.However,in the leaves transformed with NLR1-Vpro_316-ATGthere was no GUS expression cells.The results showed that the 668bp-316bp region of the promoter was the core region in response to Bgt.By analysis using Plant Care and Pl ACE websites,MYB-binding sit e,GT-1,ABRE（abscisic acid response element）and STRE（stress response element）were found in this region.The deletion one or both MYB-binding site cis-elements had no effectcs on the Bgt-responsive activity,indicating that these two elements w ere not the key cis-elements in the promoter region in response to Bgt.Others cis-e lements need to be further investigated.3.Cloning and evolutionary analysis of the promoters of the NLR1-V allelesAccording to the reference of Chinese Spring,the promoers of Ta-NLR1-A,Ta-NLR1-B and Ta-NLR1-D were cloned and inserted to pWMB002 to form the recomninant vector pWMB002:pTa-NLR1-A:GUS,pWMB002:pTa-NLR1-B:GUS,pWMB002:pTa-NLR1-D:GUS.The activity of three promoters were detected by transient expression technique,and it was found that few GUS expressed cells were detected in the Bgt inoculated leaves.At the same time,the promoters of NLR1-V alleles were cloned from60 accessions of Haynaldia villosa,and the promoters could be divided into three classes.In Class I,there was a 71bp deletion in 721bp-791bp region and a 22bp deletion in117bp-138bp region.In Class II,there was a 27bp deletion in 199bp-225bp region.In Class III,there was a fragment deletion but many SNPs.Multiple alignment indicated that the region 316bp-668bp was conserved and very few SNPs distrubuted in this region.The gene expression analysis showed that the four representative promoters could response to Bgt.