Cloning And Functional Analysis Of OsHX8 In Rice Sheath Blight Resistance

As a widespread fungal disease in the world,sheath blight occurs frequently and causes serious damage in rice.At present,due to the lack of varieties with high resistance to rice sheath blight and the special infection mechanism and living habit of Rhizoctonia solani,the research on breeding of rice sheath blight resistant varieties is slow.Breeding resistant varieties is the most effective and economical way to control rice sheath blight,so digging out more resistant genes can promote the development of disease resistance breeding.P450 gene family is a large and versatile gene family in plants,which can affect the growth,development and metabolism of plants.However,most of the annotated P450genes have not yet been cloned and functionally revealed.The advent of the genomic era has provided more convenient and abundant channels for the study of the relationship between gene inheritance and protein function.In this study,based on the transcriptome data of Rhizoctonia.solani AG 1 IA infected with Teqing（TQ）and Lemont at different time periods measured by our research group.the differentially expressed P450 genes in disease-resistant and susceptible rice were analyzed,and it was found that a lot of cytochrome P450 genes were up-regulated or down-regulated in different degrees after rice TQ and Lemont were infected.A gene OsHX8 with up-regulated expression in disease-resistant material TQ and low expression in susceptible material Lemont was screened out.After TQ infection,the gene showed up-regulated expression trend with time,and low expression before and after induction in Lemont.Through bioinformatics analysis,this gene belongs to the typical P450 gene,which contains the conserved P450 Fxx Gxxx Cx G,Exx R and Gxx R motifs.This gene also contains the transmembrane domain and has the basic signal peptide region of secreted membrane protein,indicating that the encoded protein is a typical secreted membrane protein.The RT-q PCR analysis of this gene in different tissues and organs at different growth stages showed that the expression of this gene was high in the tillering stage of rice growth,while the high expression in the stem and leaves at the tillering stage coincided with the characteristics of sheath blight.OsHX8’s transgenic lines RNA interference in TQ and overexpressed in Lemont were constructed using transgenic technology.Inoculation of in vitro leaves and living leaf sheaths in positive plants with R.solani infection showed that interference reduced TQ resistance to rice sheath blight,and overexpression enhanced Lemont resistance to rice sheath blight.RT-q PCR analysis found that JA synthesis related genes were up-regulated in over-expressed lines,suggesting that the overexpression of this gene promoted JA signaling pathway.The concentration of H₂O₂ increased after R.solani infection in over-expressed lines,indicating that the enhanced resistance of this gene was related to the outbreak of reactive oxygen species.Quantitative analysis of the genes related to plant protectin synthesis in overexpressed lines revealed up-regulated expressions of OsCPS4,OsKSL7 and OsKSL8,indicating that this gene may activate the synthetic pathway of Phytoalexins,during the interaction between R.solani and rice.Any more,to further study the OsHX8 gene,we have captured prey in yeast library by yeast-two-hybrid screening,and obtained some candidate genes.Based on the above results,it is speculated that OsHX8 is resistant to sheath blight to some extent,laying a foundation for subsequent studies on the mechanism of this gene,as well as for the utilization of transgenic genes and the screening of disease-resistant germplasm resources to promote disease-resistant breeding.