Molecular Cloning And Functional Analysis Of Hm501 From Hirschmanniella Mucronata

The rice root nematode Hirschmanniella spp.are important endoparasitic nematodes of rice,which harms more than 58% of the world’s rice fields,causing up to 25% yield loss.Jiangxi Province is a large rice-growing province,and H.mucronata is a serious harm in Jiangxi’s rice-producing areas.In order to seek safe and efficient control methods,it is vital to understand the pathogenic mechanism of H.mucronata.In order to make an intensive study of the pathogenic mechanism of H.mucronata’s infestation in rice,Transcriptome sequencing of H.mucronata which intestated resistant rice "RN51" 、susceptible rice "RN155" and H.mucronata which didn’t intestate rice were collected and examined respectively using RNA-seq in this study.10794 up-regulated differentially expressed genes and 5531 down-regulated differentially expressed genes were obtained in total.From the up-regulated differential expressed genes,Hm501 was predicted to be expressed in esophageal glands which was selected to take a molecular cloning and functional analysis.The full-length c DNA sequence of Hm501 was obtained by RT-PCR,which included a 732 bp opening reading frame and encoding 243 amino acids.Hm501 had a putative signal peptide in N-terminus and a rdx superfamily domain with CXXC active site in C-terminus,which suggested that its function may be related to the quality control of protein folding in endoplasmic reticulum.In situ hybridization analysis showed that Hm501 was expressed within the subventral esophageal gland.A plant expression vector of Hm501 was constructed for transient expression in tobacco.Subcellular localization showed that Hm501 localized in the endoplasmic reticulum and couldn’t induce allergic necrosis in Nicotiana benthamiana or suppress the programmed cell death mediated by BAX.Nematode inoculation test showed that RNAi transgenic rice could silence Hm501 expression effectively,indicating that capacity of reducing the infection ability of H.mucronata.The prokaryotic expression vector of Hm501 was constructed and the induction conditions were optimized.Optimization results showed that the best induction condition of soluble expression of Hm501 appeared in 15 ℃,0.5 m M IPTG induced for 16 h.Hm501 protein was obtained after mass expression and purification,and was incubated with rice root protein,and the pull-down test was performed to screen the interacting proteins.Multiple specific bands were obtained,which were detected by SDS-PAGE,which required to be identified through mass spectrometry.In conclusion,Hm501 plays an important role during the infection.It’s necessary to further analyze the potential interaction protein information of Hm501 protein and the mechanism of action,so as to clarify the specific functions of Hm501 in the process of nematode infection.