Cloning Of Rice Thionin-like Genes And Its Role In Resistance To Rice Root-knot Nematode Meloidogyne Graminicola

Rice root-knot nematode,Meloidogyne graminicola,is one of the most destructive plant parasitic nematodes in rice,which has been a major problem restricting rice production in China and Asia.Due to its wide host range and masked symptom,coupled with the unclear existing resistant rice varieties,and no resistance gene(s)to M.graminicola have been identified to date,the management method of planting resistant varieties has not been widely used.To disclose the resistance mechanisms of rice varieties,by resistance/susceptibility of different rice varieties to M.graminicola,genome-wide association study,bioinformatics analysis,quantitative real-time PCR,label-free proteomics analysis,overexpression genetic transformation,prokaryotic expression protein and other experimental methods were employed on 4 thionin-like genes,and the following research results were obtained.Results will provide references for nematode disease resistance and molecular genetic breeding in rice.Resistance/susceptibility of 345 rice varieties to M.graminicola were evaluated from nature population under field conditions in Hanshou,Hunan Province.The results indicated that 9 resistant,281 moderate susceptible,54 susceptible and 1 highly susceptible rice varieties showed same resistance reaction in two different years.Combined with pot experiment results,it shows that Khao Pahk Maw,IRGC6304-2 and Zhonghua 11 are resistant varieties.Genome-wide association study revealed that an approximately 4.5 Mbp segment located on chromosome 7 in rice is associated with resistance to M.graminicola.In addition to 378 genes encoding transposon-related protein and 109 genes without annotation information,a total of 175 candidate resistance genes were obtained in this segment.Previous studies have shown that thionin genes play a vital role in against M.graminicola in rice,and thionin-like genes are involved in resistance to Heterodera schachtii in Arabidopsis.Consequently,four rice thionin-like genes in this segment were selected for functional verification.Four thionin-like genes,LOC＿Os07g24820,LOC＿Os07g24830,LOC＿Os07g25050 and LOC＿Os07g25060,encode 107,95,95 and 86 amino acids,respectively,and the molecular weight of each protein is about 10 k Da.Four thionin-like proteins have signal peptides with the same cleavage site and no domains.They have homologous sequences in Oryza sativa subsp.indica and O.rufipogon,but no functional information.LOC＿Os07g24820 gene has no intron in resistant varieties.LOC＿Os07g24830and LOC＿Os07g25050 genes have multiple genetic variation sites between resistant and susceptible varieties.Quantitative real-time PCR and label-free proteomics were employed to analyze the expression profile of thionin-like genes and proteins in roots/galls.The results showing that the expression level of thionin-like genes in susceptible variety was higher than that in resistant varieties.M.graminicola induced the expression of thionin-like genes and proteins in roots/galls.Transgenic rice with over-expression thionin-like genes were obtained through genetic transformation,and its resistances to M.graminicola were evaluated using pot experiment.The results revealed that over-expression of the LOC＿Os07g24830 or LOC＿Os07g25050 gene in NPB significantly enhanced the susceptibility to M.graminicola.Over-expression of the LOC＿Os07g25060 gene in NPB had a tendency to increase the susceptibility to M.graminicola,indicating thionin-like genes negatively regulated the resistance of rice to M.graminicola.The prokaryotic expression of the protein encoded by the LOC＿Os07g24820 gene was purified and tested for its activity against M.graminicola.The results showed that there was no significant change on the mortality of nematode treated with the fusion protein compare with buffer or GST protein,indicating that the thionin-like protein expressed in prokaryotic cells had no nematicidal activity.In conclusion,this study evaluated the resistance/susceptibility of rice populations to M.graminicola,and identified Khao Pahk Maw,IRGC6304-2 and Zhonghua 11 as resistant varieties.Genome-wide association study revealed that the 4.5 Mbp segment of rice chromosome 7 is related to the resistance of M.graminicola.The four thionin-like genes and proteins contained in this segment can be induced by M.graminicola,which plays a negative role in regulating rice resistance.This study provides a theoretical basis for molecular breeding of resistant varieties and eco-friendly management of M.graminicola.