Functional Analysis Of Two Brown Planthopper Resistance Related Genes

Small Expressed Protein(s EP)genes are those annotated as expressed protein(EP)in rice genome and whose ORF less than 450 bp.EP are widely present in rice genome and get the name due to the presence of EST and FL-c DNA support which shows that EP have transcriptional and translational activity.SEP is a kind of small Open Reading Frame(s ORF),which have been neglected due to its difficulty in annotation and identification.In recent years,with the progress of sequencing technologies,a large number of s ORF with transcriptional and translational activities have been discovered,and it has been demonstrated that s ORF have an great impact on the growth and development of plants.However,the role of s EP in brown planthopper(BPH,Nilaparvata lugens St(?)l)resistance is still not clear.So we hope to explore the role and the research value of s EP in rice,in the mean time,we can also identify new genes which involve in the BPH resistance.We obtained a large number of BPH-induced s EPs through transcriptome technology previously.In this study,we selected two of them for study,and the expressions of both genes were continuously induced by BPH and showed a large fold change.We named the two s EP as BPH Resistance related Protein,OsBRP1(BPH Resistance Related Protein 1)for LOC＿Os12g05690 and OsBRP2(BPH Resistance related Protein 2)for LOC＿Os02g50110 respectively.QRT-PCR results identified these two genes expression trends which were obtained by transcriptome.We further constructed the knockout lines and overexpression lines of OsBRP1 and OsBRP2 respecetively,and these transgenic lines were further used for identifying of BPH resistance.Compared to the wild-type(WT),the resistance of OsBRP1 overexpression lines was decreased,and the resistance of OsBRP2 knockout lines was increased,while the BPH resistance of OsBRP1 knockout lines and OsBRP2 overexpression lines have no significant difference.Therefore,OsBRP1 and OsBRP2 showed the negative regulation of rice BPH resistance.What’s more,the phenotypes of OsBRP1 overexpression lines were characterized as dwarfism,enlarged leaf angles and low fertility.OsBRP2 has also been identified to control the length of rice style.Thus,OsBRP1 and OsBRP2 are not only involved in the regulation of rice growth but also in BPH resistance.What’s more,we explored the mechanism of OsBRP1 and OsBRP2 by subcellular localization analysis,cis-regulatory elements analysis,tissue expression analysis,interaction protein prediction and q RT-PCR analysis of hormone-related genes.As a result,OsBRP1 was co-located in the nucleus and cell membrane,and OsBRP2 was only located in the nucleus.The promoter of OsBRP1 and OsBRP2 were both contain a large number of tissue-specific elements,pressure response elements,hormone response elements and transcription factor action sites.The expression level of OsBRP2 in plants was higher than that of OsBRP1,and both of them highly expressed in roots,stems and leaves.OsBRP1 was not expressed in anther and stigma,while OsBRP2 was highly expressed.OsBRP1 and OsBRP2 have a large number of hormone response cis-elements,espectially JA and Me JA response cis-elements.Both OsBRP1 and OsBRP2 were predicted to interact with hormone-related genes,for example OsBRP2 was predicted that interact with several JAZ proteins.The expression of JA related genes in OsBRP1 and OsBRP2 transgenic lines were significantly different from WT.Therefore,we suggested that OsBRP1 and OsBRP2 might affect JA signaling in plantsTo sum up,OsBRP1 and OsBRP2 are both induced by the infestation of BPH,and play a role in BPH-resistance and development in rice.Their functional mechanisms need to be further explored.These results also show that some of s EP play an important role in the resistance of BPH and growth in rice,and the more functions of s EPs are worth identifying and exploring.