Cloning And Expression Analysis Of Resistance Genes Zl-RPM1 And Zl-ADR In Zizania Latifolia

The swollen stem of Zizania latifolia was caused by the interaction between Ustilagoe sculenta and plants of Zizania latifolia.The expression profile of proteome in the stems of Z.Latifolia among different stages of development showed that some proteins which associated with disease resistance and stress response in plants were found expressed differently.The immune responses of Z.Latifolia plant caused by the infection of U.esculenta could play an important role in the formation and growth of the swollen stem.The study of disease-resistant genes involved in Z.Latifolia would provide theoretical basis for pregnant mechanism.In this study,the Long jiao2 Z.latifolia of two-season cultivar was used as the research material.Full-length sequences of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR genes were cloned in Z.Latifolia and sequences were analysised by bioinformatics.At the same time,the expressions of the three genes were proceed of during the process of development,different types and parts of Z.Latifolia.Based on the observing of tissue slice,the growth and distribution of U.esculenta were analyzed during the three types of Z.Latifolia.In addition,the growth of U.esculenta was inhibited after triadimefon processing,the expression of the three resistance genes were associated with the growth and distribution of U.esculenta.And the expression changes of the three disease-resistant genes were verified through U.esculenta interaction with Z.Latifolia callus.The results were as follows:1.Observation of the infection and distribution of U.esculenta in the development of Z.LatifoliaThe distribution of U.esculenta existed obviously differences in different phenotype Z.Latifolia.The stem of normal Z.Latifolia existed a lot of spores when it becamed older,which was similar to the grey Z.Latifolia.In the process of intumescence stem,the numbers of hypha cluster were the maximum,at 10 cm and15 cm,but decreased significantly at 20 cm.It might be related to the lack of nutrition when the Z.Latifolia plant becamed older.2.Cloning and sequences analysis of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR in Z.LatifoliaFull-length cDNA sequences of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR were obtained by RACE.Sequence homology analysis showed that,Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR were highly homologous with Oryza sativa,and were respectively of 61%,79% and 95%.And they all had conservative structure domains,such as CC,NBS,LRR and so on.The Zl-RPM1.1 gene has a cDNA full length of1665 bp,containing an open reading frame of 1593 bp,encoding 531 amino acids.It was predicted that molecular weight was approximate 45.78 kDa,and theoretical isoelectric point was 8.29.The Zl-RPM1.2 gene has a cDNA full length of 3480 bp,containing an open reading frame of 3195 bp,encoding 1028 amino acids.It was predicted that molecular weight was approximate 117.8 kDa,and theoretical isoelectric point was 6.30.The Zl-ADR gene has a cDNA full length of 1164 bp,containing an open reading frame of 1161 bp,encoding 387 amino acids.It was predicted that molecular weight was approximate 44.10 kDa,and theoretical isoelectric point was 6.43.Through the Spidey program of NCBI,we found that only Zl-ADR gene contained introns,from 629 bp to718 bp and from 931 bp to 1315 bp,were respectively 90 bp and 185 bp.3.The expression pattern analysis of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR genes from Z.LatifoliaThe response time was different of three resistance genes during stem development in Z.Latifolia.The expressions of Zl-RPM1.1 and Zl-RPM1.2 genes were significantly increased at 8 leaves stage and 15 cm,and the expression of Zl-ADR increased at 8 leaves stage,but decreased at 15 cm.The 8 leaves stage of Z.Latifolia might be an important period of immune response,which might be associated with U.esculenta infection.The expressions of the three genes were various in different parts of Z.Latifolia.The expressions of Zl-RPM1.1 gene in the leaves were significantly higher than the stem in normally swollen Z.Latifolia.The expressions of Zl-RPM1.2 and Zl-ADR gene in the stem were significantly higher than the leaves both in normally swollen and non-swollen Z.Latifolia.In addition,the expressions of three genes were obvious during different phenotypes Z.Latifolia.The expressions of Zl-RPM1.1 and Zl-RPM1.2 genes in grey Z.Latifoliawere significantly higher than non-swollen stem and normally swollen stem in Z.Latifolia at initial stage of the swollen.The expression of Zl-ADR gene in normally swollen stem Z.Latifolia were significantly higher than non-swollen and grey Z.Latifolia at initial stage of the swollen.It might be related to the infection ability and the mode of nutrition of U.esculenta.Z.Latifolia gestation could be significantly delayed after spraying triadimefon at8 leaves stage,and it was existed dose effect within a certain range.The triadimefon could obviously inhibit the hypha growth of U.esculenta through observation of tissue slice.The swollen stem in Z.Latifolia plant was closely related to the growth and distribution of U.esculenta.The results of fluorescence quantitative PCR showed that,the expressions of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR genes were all increased in the triadimefon of 20 mg/L.However,the expressions of the three genes were all significantly reduced in the triadimefon of 80 mg/L.Swollen stem of Z.Latifolia and the expressions changes of the three resistence genes were closely related to the infection and growth of U.esculenta.The results of U.esculenta interaction with Z.Latifolia callus showed that,The expressions of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR genes were significantly changed after both MT type and T type U.esculenta interaction with Z.Latifolia callus.But T type U.esculenta,which caused the three resistance genes of response time,was earlier than MT type.Compared with the controls,the expressions of Zl-RPM1.1,Zl-RPM1.2 and Zl-ADR genes were significantly reduced both in the leaves and the stems,after Helminthosporium zizamae Nishik or Uromyces coronatus Miyabe et Nishuida infection.