| Brown-spot of tobacco caused by Alternaria alternate is a fungal disease that widely spreading across the world,its character are fast spreading and difficulty to cure.After pathogen infecting tobacco leaves,the integrality of leaves can be destructed and chemical components of leaves are change,all these seriously lower the quality of tobacco leaves,then lead to a great commercial loss.Using chemical drugs to defense brown-spot not only pollute the environment,but also influent the quality of tobacco.At present the most practical way to solve this problem is developing resistant cultivar.The tobacco resources with high resistance to brown-spot is very limited,fully excavating the existing resistant resources to improve the resistance of tobacco is an important topic of the tobacco resistance breeding.The response to disease of tobacco is a complex physiology and pathological process,involved of a series of genetic factors and functional genes,taking advantage of modern molecular techniques,revealing the response mechanism of tobacco resistance to disease from molecular level is of great significance to tobacco resistance breeding.At present studies of phytohormone signal transduction and the regulating expression of key transcription factors and target genes in it have being a hot point content in the area of plant molecular stress resistance breeding.The main phytohormone like jasmonic acid,ethylene,and salicylic acid play an important role in plant disease response,through mutual synergistic or antagonistic effects,different signal pathways format a complex and orderly phytohormone regulation network.The pathogen infection can activate the synthesis of phytohormone,then induce the expression of downstream defense genes like pathogenesis-related genes(PR genes),thereby enhance the disease resistance of plants.Phytohormone activating expression of PR proteins needs a series of regulator proteins,in the upstream of phytohormone signal pathway kinase and transcription factors give an of great affection on the expression and regulation of PR proteins,and this is a key factor to the disease resistance of plant.As a high quality tobacco cultivar,K326 is widely planted in China,through comparing the molecular difference between Beinhart 1000-1 and K326,excavating resistance marker to brown-spot,and developing the resistant breeding of K326 will promise well.In this study through the physiological and molecular biology technology,the disease resistant difference between the high resistance cultivar(Beinhart 1000-1)and the susceptible one(K326)to brown-spot is firstly analyzed and compared.After being infected with brown-spot pathogen,the leaves of Beinhart 1000-1 appeared local tissue necrosis,but the disease spot did not spread,and presented a highly resistance to brown-spot.But after K326 being inoculated with pathogen,leaves did not appear local tissue necrosis,actually its disease spot will persistently spread until all rotted,presented to be highly susceptive to brown-spot.Further using qRT-PCR technique,this study analyzed the expression difference of 11 PRgenes and 16 transcription factors belonged to three families(WRKY,bHLH and AP2 /EREBP)inresponsing to pathogen and phytohormone(jasmonic acid and ethylene)in Beinhart 1000-1 andK326,revealed the molecular basis of resistance differences.Results showed that:1.PR genes and transcription factors show obviously different level of sensitivity to the pathogen of brown spot respectively in Beinhart 1000-1 and K326Through comparing the relative expressional level of related genes before and after tobacco being inoculated with pathogen,results showed that 9 of the eleven PR genes present a higher sensitivity to pathogen in Beinhart 1000-1,as they were PR1 a,PRB-1b,PR3 a,PRQ,PRR,PR4,PR5,PR3 b and Gluc;13 of sixteen transcription factors respectively belonged to AP2 /EREBP,bHLH and WRKY families presented a higher sensitivity to pathogen in Beinhart 1000-1,as they were ERF1,ERF2,ERF3 b,ERF8,ERF32,ERF168,EREBP1,CEF1,XP1,TSi1,WRKY4,WRKY11 and MYC2.2.PR genes and transcription factors show obviously different level of sensitivity to JA and ET respectively in Beinhart1000-1 and K326Through comparing the relative expressional level of related genes before and after tobacco being treated with JA,results showed that in PR genes,PRB-1b,PR5,Gluc and PRR presented a higher sensitivity to JA especially in Beinhart 1000-1;As for transcription factors,ERF8,TSi1,XP1,WRKY11 and MYC2 presented a higher sensitivity to JA especially in Beinhart 1000-1.Through comparing the relative expressional level of related genes before and after tobacco being treated with ET,results showed that in PR genes,PRB-1b,PR5,Gluc and PR3 b presented a higher sensitivity to ET especially in Beinhart 1000-1;As for transcription factors,ERF8,ERF168 and WRKY11 presented higher a sensitivity to ET especially in Beinhart 1000-1.Results also showed that JA,ET and pathogen could easier induce the expression of PR5,Gluc and PRB-1b as well as their upstream transcription factors,WRKY11 and ERF8 in Beinhart 1000-1,but K326 did not showed the same situation,revealing the possible resistance mechanism of tobacco: after tobacco being violated by brown-spot pathogen,the synthesis of JA and ET activated the JA and ET signal transduction pathways,then related transcription factors like WRKY11/ERF8 could further regulate and activate the expression of downstream target genes like PR5/Gluc/PRB-1b,eventually plants generated the resistance to brown-spot.In addition,as a key transcription factor in the JA signal pathway,MYC2 was induced by pathogen of brown-spot and JA,and its absolute expression level in Beinhart 1000-1 was fairly higher than that in K326,proved that JA signal pathway may play a leading role in the immune response of Beinhart 1000-1 defending brown-spot.Seeds germination and seedlings growth and decelopment tests further proved that Beinhart 1000-1 was quitely sensitive to JA.JA could not only strongly inhibit Beinhart 1000-1 seeds germinating,but also inhibit leaves and roots of seedlings growing and developing,while this phenomenon was not obvious in K326.All these results proved that JA signal pathway played an important role in regulating tobacco defending brown-spot. |
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