Cloning And Characterization Of GT1 Genes From Wheat And Rye

Fusarium head blight(FHB),also called scab,caused mainly by Fusarium gramearum,is one of the most severe worldwide diseases resulted in yield loss and deterioration of seed quality of wheat.In addition,mycotoxins contained in the infected grains were very harmful to both human and animals.Development of wheat cultivars with low toxins is the most effective way to control this disease.At present,at least three toxins including deoxynivalenol(DON),3-ADON and zearalenone(ZEN) were isolated and identified from the diseased wheat.These toxins could not only inhibit the biosynthesis of eukaryotic proteins,but also interfere the expression of plant defense genes.Of them,DON is regarded as one of the most important pathogenic factors in plant.However,little is known about the expressed patterns of wheat genes induced by DON,and no DON resistance genes were isolated from wheat.In this paper,based on the Affymetrix wheat gene chips contained 55,052 transcripts,950 up-and 165 down-regulated ESTs,including 6 up-regulated genes encoded glucosyltransferase(GT) were identified from the well known scab-resistant wheat landrace Wangshuibai induced by DON.Two EST genes BQ281752 and BJ250503 were confirmed as the up-regulated genes expressed in Wangshuibai after analyzed by RT-PCR,both genes showed the maximum level at 24 h after inoculated with DON by single floret injection (SFI).Using a set of Chinese Spring nullisomic/tetrasomic lines,the two genes were mapped on chromosome 5D and 2D,respectively.Amplified with 3'-RACE PCR,three paralogous sequences of Wangshuibai were isolated and cloned,which were preliminarily designated as NAU-1,NAU-2 and NAU-3,respectively.Of them,NAU-1 and NAU-2 showed 91%and 92%identity to BQ281752,while NAU-3 with 94%identity to BJ250503.Searched with the DON degeneration gene DOGT1 of Arabidopsis thaliana,a full-length GT gene(BT009372) from Chinese Spring was identified from current wheat ESTs.Gene specific primer was designed and three full-length cDNA were isolated from Wangshuibai, Alondra'S and Mianyang 85-45,which were preliminarily designated as NAU-4(1673 bp), NAU-5(1673 bp) and NAU-6(1677 bp),respectively.In comparison with the sequences of BT009372,99%nucleotides were common between the four wheat genotyoes,while 1% nucleotides showed polymorphism.Of them,most mutants were base transition.Analyzed with RT-PCR,NAU-4 from Wangshuibai was found to express in root,leaf,stem,and spikes of wheat,while it was not induced by DON,indicating constitutive expression of this gene.The full-length gDNA sequences of NAU-4,NAU-5 and NAU-6 were isolated and cloned from Wangshuibai,Alondra'S and Mianyang85-45 with the specific primer of BT009372, which were preliminarily designated as NAU-7(2002 bp),NAU-8(2001 bp) and NAU-9 (2006 bp),respectively.After amplified in rye,barley,Roegneria kamoji and Leymus racemosus with this primer,only one gene from rye designated NAU-10(1963 bp) was successfully isolated,indicating the closer relationship between wheat and rye.Different from Arabidopsis thaliana with only one exon in this gene(no intron),2 exons and 1 intron were discovered in wheat,rye and flee.The comparison result also revealed that the exon of this gene conserved well while the intron changed rapidly.Phylogenetic analysis showed that wheat GT1 genes were closer to rye and rice,then to Arabidopsis thaliana,Vitis vinifera and Nicotiana tabacum etc.The further cluster with GT1 family 1 genes of Arabidopsis thaliana,the GT1 genes from wheat and rye were grouped into 73C subfamily. Prokaryotic expression vector pET32a-NAU-4 was constructed.The recombinant plasmid was transformed into Escherichia coli and could be stably expressed in BL21.The maximum expression level of His-NAU-4 was obtained when the concentration of ITPG, the inducing temperature and the inducing time were at 0.5 mmol/L,28℃and 6 h, respectively.The result showed that NAU-4 was an active gene isolated from Wangshuibai. Additionally,both the sense and antisense expression vectors of Agrobatrium plasmid (132-NAU-4a and 132-NAU-4) were also constructed.