Cloning And Functional Analysis Of NPR1 Gene In Zizania Latifolia

The interaction of Zizania latifolia and Ustilago esculenta play an important role in the formation of swollen stem.U.esculenta grew and spreaded in the stem of Z.latifolia as a biotrophic pathogenic fungi.As a matter of fact,the swollen stem was a disease phenomenon of Z.latifolia plant caused by infection of U.esculenta.The results of the previous study had showed that the expression of resistance genes were related to the growth and distribution of U.esculenta.NPR1 gene was one of the key genes in plant defense against pathogenic infection which was closely related to the infection of biotrophic infection.Full length squences of Zl-NPR1 、 Zl-TGA and Zl-PR1 were cloned in this study.The interaction between Zl-NPR1 and Zl-TGA were analyzed and the expression patterns of Zl-NPR1 、 Zl-TGA and Zl-PR1 genes were studied among different Z.latifolia pregnant phynotypes during the formation and development of the swollen stem.The regulation pattern of the related genes were discussed as the infection and distribution of Ustilago esculenta,which provided the theoretical basis for the study of the mechanism of the interaction between the Z.latifolia and U.esculenta.The results are as follows: 1.Full length cloning of Zl-NPR1,Zl-TGA and Zl-PR1 genes in Z.latifoliaThe full length of Zl-NPR1 gene was obtained with a total length of 1731 bp,encoding 576 amino acids,and the genomic DNA was 4310 bp as well as contains four exons and three introns.CDD prediction showed that the Zl-NPR1 contained the BTB domain,ANK repeat domain and NPR1 like C-terminal domain,belonging to NPR1 gene.Blast analysis showed that it was close to Oryza brachyantha with 88% high identity.Phylogenetic tree analysis showed that Zl-NPR1 was associated with Oryza brachyantha and Oryza sativa which were clustered into one class.The full-length cDNA sequences of Zl-TGA2,Zl-TGA4 and Zl-TGA6 were obtained.Zl-TGA2 cDNA has 1604 bp,encoding 517 amino acids.Zl-TGA4 cDNA has 1305 bp,encoding 434 amino acids.Zl-TGA6 cDNA has 1455 bp,encoding 463 amino acids.CDD predicted that the Zl-TGA2/Zl-TGA4/Zl-TGA6 contains the bZIP domain and DOG1 domain.Phylogenetic tree analysis showed that Zl-TGA4 belongs toⅠtype TGA,which were clustered into one class with Oryza brachyantha TGA4.Zl-TGA2 and Zl-TGA6 belong toⅡ type TGA.And it was in close relationship with Oryza sativa,Oryza brachyantha,Brachypodium distachyon.The full length cDNA sequences of Zl-PR1 was 1604 bp,encoding 517 amino acids.CDD predicted that the Zl-PR1 contains the SCP PR-1like domain,belongs to PR1 gene.Blast analysis showed that it was close to Oryza brachyantha with 89% high identity.Phylogenetic tree analysis showed that Zl-PR1 was associated with Oryza sativa’ homologous gene which were was clustered into one class.2.Analysis of interaction between Zl-NPR1 with Zl-TGA2,Zl-TGA4 and Zl-TGA6With the method of PEG/LiAc transformation,pGBKT7-NPR1 recombinant plasmid was transformed into Y2 HGold yeast strains,respectively transforming pGADT7-TGA2,pGADT7-TGA4 recombinant plasmid into yeast strain Y187.Yeast two hybrid analysis showed that blue colonies were formed after pGBKT7-NPR1 and pGADT7-TGA2,pGBKT7-NPR1 and pGADT7-TGA4 were co-cultured on SD four-deficient medium,but the colonies were white when pGBKT7-NPR1 and pGADT7-TGA6 were co-cultured.The results showed that Zl-NPR1 could interact directly with Zl-TGA2 and Zl-TGA6 transcription factors,but it could not directly interact with Zl-TGA4.3.Analysis of the expression pattern of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes in the Zizania latifoliaThe response time of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes were different in the formation and growth of the swollen stem in three pregnant phenotypes of Z.latifolia.In normal Z.latifolia,the expression of Zl-NPR1,Zl-PR1,Zl-TGA2 and Zl-TGA6 genes were mainly in 8 leaves stage and the stems expanded to 10-15 cm,but the expression of Zl-TGA4 gene was not obvious.In grey Z.latifolia,the expression of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes were mainly increased at 5 leaves stage and the stem expanded to 5 cm.In male Z.latifolia,the expression levels of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes were significantly up-regulated in the initial expansion.This fact indicated that the infection ability of U.esculenta in different pregnant phenotype are different.In addition,the expression level of Zl-NPR1 in leaves and stems was significantly different in normal Z.latifolia.And the expression of Zl-NPR1 was higher than that of leaves,which was closely related to the distribution of Ustilago esculenta,indicating that the expression of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 could be related to Z.latifolia pregnant phenotype,the infection ability of U.esculenta and the biotrophic pathogenic bacteria.Spraying triadimefon could inhibit the growth of U.esculenta and regulate expansion of Z.latifolia stem,and the expression of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 were down-regulated in the early stage(sprayed after 7 days)after treatment with triadimefon,which was related to the inhibition of the growth and distribution of U.esculenta in the stem.The expression of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes increased gradually with the prolonging of treatment time,which were related to the recovery of growth and distribution of U.esculenta and changes of stem expansion.The results showed that the expression of Zl-NPR1,Zl-TGA2,Zl-TGA4,Zl-TGA6 and Zl-PR1 genes in normal Z.latifolia was regulated by the infection,growth and distribution of U.esculenta as well as stem expansion.