| Meloidogyne graminicola is one of the most important pathogens of rice root-knot nematode,which seriously affecting rice yield and widely distributed in Myanmar,Bangladesh,Laos,Thailand,Vietnam,India,China,the Philippines and the United States,et al.In recent years,it has become a major rice root disease that restricts rice quality and yield,the occurrence area of rice root-knot nematode disease is expanding in major rice growing areas of China.However,functional genomics of the basal molecular biology in M.graminicola had been progressed rather slowly and even rarer compared to Caenorhabditis elegans,other plant parasitic nematodes such as M.incognita and M.hapla.In this study,the rice root-knot nematodes disease was identified,and four flp genes of M.graminicola were cloned,the function of Mg-flp-14 and Mg-flp-18 genes were analyzed by in situ hybridization and RNAi.The important morphological characteristics of two root-knot nematode populations(1MSY and 10MDZ)collected from the rice field in Hainan were observed by measuring and the perineal pattern of traditional morphological identification method,which was identified as M.graminicola.28S rDNA-D2/D3 region sequence from nematode sample was validated by the biological technique.The results of homology analysis showed that the populations(1MSY and 10MDZ)of the root-knot nematodes were higher homologous when compare with similar species in the same species or genus,and lower homologous when compare to the different species in different genera.The pathogenic nematodes(1MSY and 10MDZ)of rice root-knot nematode collecting from the two places were identified as M.graminicola combining with the results of morphological identification,molecular biological identification and homology analysis.The fragments of four flp genes in M.graminicola,Mg-flp-1(GenBank Accession No.MK886770)、Mg-flp-13(GenBank Accession No.MK886771)、Mg-flp-14、Mg-flp-18,were cloned by RT-PCR.The fragment sizes were 493 bp,269 bp,673 bp and 721 bp,respectively.The full-length 1,047 bp of Mg-flp-14(GenBank Accession No.MK855119)and 931 bp of the 3’ end of Mg-flp-18 gene(GenBank Accession No.MK886769)were obtained by RACE.The Mg-flp-14 gene contains a 315 bp open reading frame(ORF)encoding 104 amino acids,a 5’ untranslated region of 644 bp,a 3’ untranslated region of 88 bp,and an oligo Adenine tailing signal(aataaa)at the 3’ end;The 3’ end of the Mg-flp-18 fragment has a 408 bp ORF encoding 135 amino acids,3’ untranslated region 352 bp,and the 3’ end contains an oligo Adenine tailing signal(aataaa).The proteins structure encoded by Mg-flp-14 and Mg-flp-18 were analyzed.The location analysis of Mg-flp-14 and Mg-flp-18 in M graminicola by in situ hybridization.The results showed that Mg-flp-14 was prominently expressed in the nematode stylet,pharyngeal nerve ring and nerve ring around the excretory hole,and there was partial dispersion in the middle of nematode;significantly expressed of Mg-flp-18 in the nerve ring around the worm excretory hole,there is also partial dispersion in the middle of nematode.The silencing effects of Mg-flp-14 and Mg-flp-18 were examined by RNAi.In vitro immersion study,the results of M.graminicola J2 resorcinol concentration and soaking time screening showed that 0.3%resorcinol stimulation for more than 6 h was more favorable for vitro phagocytosis of M.graminicola J2.Injecting gfp dsRNA into nematodes as the control and injecting Mg-flp-14 dsRNA and Mg-flp-18 dsRNA into nematodes as the treatment,semi-quantitative technique was used to detect the silencing effect of Mg-flp-14 and Mg-flp-18 in M graminicola J2,there was no silence effect on injecting Mg-flp-14 dsRNA,and a few silence effect on injecting Mg-flp-18 dsRNA. |
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