Functional Study Of The Pgr07060 Gene In Pyrenophora Graminea

Barley leaf stripe is one of the most serious diseases in barley,which causes yield loss of more than 70% in the year with crop being seriously infected.The disease is caused by Pyrenophora graminea.Research on the pathogenic mechanism of barley leaf stripe can provide a theoretical basis for the prevention of barley leaf stripe and the breeding of new varieties resistant to this disease.In this study,the functional study of the gene Pgr07060 from strong pathogenicity wild strain QWC was carried out.Results were as following:1.The gene Pgr07060 was cloned from Pyrenophora graminea isolate QWC,and bioinformatics analysis showed that the full length of the Open Reading Frame of the Pgr07060 was 1119 bp,encoding 372 amino acids,the number of Thr was the most,reached11.80%.According to the secondary structure of the protein encoded by Pgr07060,random coil accounted for 56.18%.The instability index of the protein encoded by Pgr07060 was51.96,indicating that the protein was unstable and hydrophilic.In addition,the protein encoded by Pgr07060 was a transmembrane protein.2.The transient expression vector PVX:Pgr07060 and subcellular localization vector PCAMBIA1300-35S-GFP:Pgr07060 were constructed and then transferred into agrobacterium tumefaciens GV3101.The tobacoo leaf was infected to conduct the transient expression of Pgr07060,the PVX: BAX,PVX:INF1 and PGR107 were used as controls.The results showed that PGR107 could not cause cell necrosis of tobacco leaves,but PVX: BAX,PVX: INF1 and Pgr07060 could cause severe shrinkage and necrosis in tobacco leaves,which indicated that Pgr07060 could induce cell necrosis and induce immune response.The expression of Pgr07060 was predicated in nucleus using software Cell-PLoc 2.0,the NLS sequence of this gene was PPSASTKTKKKKKKSTP.The subcellular localization results showed that the green fluorescence of Pgr07060 has a high degree of coincidence with the nuclear autofluorescence,and the membrane fluorescence was also very strong.It indicated that the expression of Pgr07060 maybe on the nucleus and cell membrane.3.The interference vector p Silent-1:Pgr07060 and the overexpression vector p BARGPE1:Pgr07060 of Pgr07060 were successfully constructed and transformed by PEG4000 mediated method.There were 3 interfering strains and 2 overexpression strains that were obtained by combining hygromycin resistance screening and q RT-PCR analysis.Compared with the wild strain,the expression of Pgr07060 in transformed strains and wild strain QWC showed that the gene expression of 3 interfering transformants decreased by45.17%,65.38% and 68.09%,while the gene expression of 2 overexpression strains increased by 668.88% and 908.79%,respectively.By measuring the colony diameter for 7 d,the colony growth rates of the 3 interfering transformants decreased by 26.91%,48.19% and 52.21%,while the growth rates of the 2 overexpression transformants increased by 61.02% and 95.76%(P<0.05),respectively.The mycelium morphology of wild strain was long and smooth,the mycelium growth was normal,while the mycelium of the interference and overexpression transformed strains were obviously separated,and the angle between the mycelium was smaller than that of the wild strain QWC.In the pathogenicity potted experiment,the leaf of Ganpi 2 was no symptoms of this disease when Ganpi 2 infected by the wild strain QWC and3 interfering strains.After infection of Alexis by the wild strain QWC and 3 interfering strains,the incidence rate of 3 interference strains decreased by 52.22%,49.29% and 49.04%(P<0.05),respectively.The barley infected by QWC grew weakly,and the leaves were obviously atrophied,and there were large areas of disease spots on the leaves,and the disease was more serious.The leaves infected by the interference strain were significantly milder,yellow-green,and there were less disease spots.