Stress-transcriptom Sequencing And Cloning And Identification Of NtRLK2 An Anti-bacterial Wilt Related Gene In Nicotiana Tabacum

Tobacco(Nicotiana tabacum L.),a member of the agriculturally crucial Solanaceae,has been extensively studied.This is because of both its economic importance and a convenient plant system for research.However,tobacco often suffers from many biotic and abiotic stresses during cultivation process,such as bacterial wilt desease in southern China,which seriously affect the yield and quality of tobacco.With the rapid development of molecular biology,biotechnology has demonstated as a powerful method in enhancing varieties with high yield,quality and stress tolerances.Although the tobacco genome sequencing project and large scale transcriptome sequencing is processed actively,only a few genes have been identified so far..In this study,a large amount of data had been obtained from tobacco transcriptome sequencing under biotic and abiotic stresses using Roche 454 pyrosequencing,then a number of stress-related genes were filtered from the transtriptome,after assembling,feature analysis and functional annotation.Based on our obtained unigene library from the transcriptomes sequencing and by merging with all the uni-EST available on GenBank,we construct a large-scale and high-density gene chip,which was used to analyze the differential transcriptome induced by the challenge of Ralstonia solanacearum,we cloned a predictive resistance gene(NtRLK2)and identified its resistance function to bacterial wilt.1.We created two normalized cDNA pools,one from RNAs of tobacco leaf stressed by drought,low temperature stress and hormone treatments,such as abscisic acid(ABA),ethylene(ETH)and salicylic acid(SA),and another from the corresponding controls.Pyrosequencing of the cDNA pools on 454 XLR70 Titanium platform produced 309,903 and 290,953 reads,respectively,with a total of 600,856 raw reads after removing low quality sequences,which were assembled to 39,912 uni genes with 16,232 unpublished genes in tobacco.Then,34 predicted stress-related genes in tobacco resistance were selected,which expression levels were validated under various biotic and abiotic conditions by RT-PCR.Four differentially expressed genes were singled out from these validated genes,and their expression pattens in tissues and during different periods under various stresseswere characterized,which showed that they were stress-related genes.2.By analyzing gene expression profiles induced by bacterial wilt,we got a fragment of a candidate gene(NtRLK2)of receptor-like protein kinase(RLK),and cloned the full-length gene from Nicotiana tabacum L.cv.CB-1.Cloning its homologous gene from resistant varieties and then building a over-expression vector and RNA interference vector which were transformed into CB-1 mediated by A.tumefaciens,we found that transgenic plants had a higher survival rate than non-transgenic plants inoculated with R.solanacearum.Among them,the resistance of the transgenic plants with RNA interference is much significant,even to higher than NtRLK2 gene over-expression transgenic plants.Accordingly,we hypothesized that there was a high-resistance gene in susceptible varieties(CB-1)and NtRLK2 functions as a suppressor of it.