Cloning And Analysis Of NBS-type Resistance Genes In Sesame Against Macrophomina Phaseolina Pathogen

Sesame is one of the important oil crops.Charcoal is a devastating fungal disease in sesame.Beside chemical control,planting disease-resistant cultivars is a safe and effective measure in sesame disease control system.Few disease-resistant cultivars can be used in sesame practice.So it’s very important to clone the disease-resistant genes and study the resistance mechanism for speeding up the disease-resistance breeding of sesame.In this study,the specific primers were designed based on NBS-RGAs blastn of charcoal-resistant cultivars with sesame EST sequences,then the disease-related genes in the resistant sesame cultivars were cloned.High resistant cultivar Shangshuinongjiazhong and susceptible cultivars JI9014,Heshangtou were chosen. The expression patterns in these cultivars with inoculation treatment and non-inoculation treatment were investigated by qRT-PCR,and the results were followed.1. Cloning of resistance-related genes in sesameTwenty-one resistance genes(GenBank accession number:KC477692-KC477707,KC771241-KC771243)have been obtained from eleven different resistance cultivars against charcoal(Macrophomina phaseolina).All of these gene sequences contained the conserved specific domains of P-loop and NB-ARC in the NBS type resistance genes encoding NBS proteins and shared the amino acid identities of35%-52%with NBS-LRR resistance disease protein and NB-ARC protein according to Blastx analyses.They were divided into four types by MEGA4.0analysis,named with SIRGA1, SIRGA2,SIRGA8and SIRGA12.2.Expression analysis by real-time PCRExpression patterns of these genes in the compatible and incompatible interactions during72h were analyzed by real-time PCR.The results indicated that the expression of SIRGA6in leaf tissue was induced in compatible interactions,while the others were induced in both interactions.The expression of SIRGA1,4,11and12were higher in the compatible interactions,while SIRGA2,8were higher in the income- patible interactions,there was no difference in SIRGA7.The expression of SIRGA1,2,4,11,12in stem tissue were all induced by Macrophomina phaseolina,and it was higher in compatible interactions.In addition,the expression of SIRGA6,8were higher in incompatible interactions.SIRGAl,4,11,12were up-regulated in both interactions.These results suggested that these genes may regulate the defense response in sesame against Macrophomina phaseolina,SIRGA8was higher in incompatible interaction,this result suggested that it may mediate the resistance defense.3.Subcellular localizationSubcellular localization of SIRGA8was observed by particle bombardment-mediated transient expression. SIR.GA8protein was revealed that it located in the cell membrane and plastid of onion epidermal cells with a laser scanning confocal microscope.lt provided a reference for the further study of the specific function of SIRGA8.