Functional Analysis Of OsGWD1 In Interaction Between Rice And Hirschmanniella Mucronata

Rice parasitic nematode is one of the most important pathogens.There are no obviously identifiable above-ground symptoms of nematode damage in the field.Retardation of growth rate occurs,especially in early growth,with a decrease in tillering.Yellowing of rice plants is observed occasionally.Because of the invadation of Hirschmanniella spp.,roots turn yellowish-brown and rotten.It is estimated that Hirschmanniella spp.infest 58% of the world's ricefields,causing 25% yield losses.Hirschmanniella mucronata is a kind of root-endoparasitic nematode,which parasitizes rice roots and secretes a variety of effector proteins to degrade or modify the rice root cell wall and alter proteins function.At the same time,rice will actively regulate the metabolism level,express cell wall modification enzyme and defense gene to resist the infection of nematodes.In previous experiment,we analyzed and compared the transcriptome results of the infected and control.The OsGWD1 gene was selected from significantly differential expression genes,cloned the gene and expressed the encoded protein to find out the interaction protein.This study is helpful in further deepening the interaction between rice and Hirschmanniella mucronata.Therefore,we carried out the following research.1.Cloned OsGWD1 gene and transformed it into pEASY-T3 vector by TA cloning technology,and the recombinant vector T3: OsGWD1 was obtained.The bioinformatics website predictive analysis showed that the open reading frame of OsGWD1 gene is 462 bp,encoding 153 amino acids.The molecular weight of the protein is about 18 KD,and the isoelectric point(pI)is 4.83.It's a hydrophilic and unstable protein.The protein has no signal peptide and does not contain transmembrane structure,which means it may not belong to the membrane protein or secreted protein.The proportion of random coils in the peptide chain is high,with 7?-sheet structures.It has a highly conserved WD40-repeat domain,belonging to WD40-repeat family.It may have 10 proteins interacting with OsGWD1,and simulats the network diagram of the interaction.2.Obtained the overexpression vector pCAMBIA1302:OsGWD1:GFP by cloning and transforming.Subcellular positioning observed that the OsGWD1 was localized in the nucleus.Obtained recombined RNAi vector pB7 G by Gateway technology.The overexpressed recombinant plasmid and RNAi plasmid were transfected into rice Nipponbrare by agrobacterium GV3101 to obtain transgenic rice plants and it provides raw materials for subsequent rice root nematode inoculation experiments.3.The orf sequence of OsGWD1 was cloned and recombined into pCzn1 prokaryotic expression vector.The positive recombinant plasmid was extracted into BL21(DE3)competent cells containing the molecular chaperone pTf16 andco-expressed with the chaperone protein.The 18 KD target protein was successfully expressed through SDS-PAGE analysis and Western Blot verification.At the 0.05 mM of IPTG,inducing 20 hours at 16 ? was the optimal expression condition which the target protein was induced and purified by Ni-NTA resin.4.We obtained 25 possiblely interaction proteins through his-tag pull down method.GO functional analysis enrichment in 20 go term and KEGG enrichment analysis showed that these proteins were enriched in 12 pathways.They were mainly concentrated in metabolic pathways such as ribosomes,oxidative phosphorylation and phagocytosis.Among them,glyceraldehyde-3-phosphate dehydrogenase,S-adenosylmethionine transferase,heat shock family protein Hsp70,Hsp73 and elongation factor alpha protein eEF1 A may play key roles in the metabolic pathway,and they have a great significance of rice in stress response and defense response.