High-throughput Screening And Functional Analysis Of Drought Resistance Genes Through RNA-seq In Brassica Napus

Brasicca napus L.is a dicotyledonous plant belonging Brassica in the family cruiferous.Brasicca napus L.is one of the most important oil crops in our country.However,with the global warming,drought as a major abiotic factor that limits crop production.Drought will have a harmful effect on the plant's growth to some extent at the whole stage of growth and development of Brasicca napus L..Drought has become a key factor of restricting high yields of Brasicca napus.Studies have shown that under abiotic stress such as drought,thousands of genes expression in plant cells have undergone changes.Some of these genes play an important role in drought tolerance mechanism in plants.Therefore,raising drought tolerance of Brasicca napus L has become the key problems to be solved immediately in agricultural development.It can be solved by making a gene that functions in drought tolerance transformed in to Brasicca napus L to cultivate drought-resistant plant germplasm resources with a genetic engineering technique.Mining for drought-enduring gene sources is an important work for Brassica napus L.resistance breeding.Identification of the genes responsible for drought-tolerance by monitoring changes in gene expression at the transcriptional level is an effective method to mine drought-tolerance genes.Transcriptome analysis can reveal the organism's gene expressing level,structural variation,discovery of new genes.In this study,preliminary tests screened drought and drought-sensitive two rapeseed cultivars were drought stress,then use Illumina HiseqTM 2000 platform for RNA collected from high-throughput sequencing,bioinformatics analysis of rapeseed transcript data,screening of the expression of genes differentially,full-length cDNA clones of these genes,and we also constructed an expression library through high-throughput Gateway technology.Expression library introduced into Arabidopsis by FOX hunting system,which constructed a mutant library of Arobidopsis which overexpress the Brassica stress resistance related genes.The main results and conclusions are as follows:(1)We applied RNA sequencing technology to characterize drought stress transcriptome of rapeseed.The statistics of the RNA-Seq reads is shown a total of 107,297 Unigenes were generated by data analysis.These unigenes were used to mine potential microsatellites,a total of 22,414 potential EST-SSRs were identified,of which 3,464 sequences contained more than 1 EST-SSR,and 1,425 EST-SSRs were present in compound form.(2)Functional classification by GO and COG,75,278 sequences can be categorized into 57 functional groups.Functional classification by KEGG showed 46,861 of 107,294 Unigenes had significant matches and were assigned to 128 KEGG pathways.The pathways with most representation by the unique sequences were metabolic pathways,biosynthesis of secondary metabolites,plant-pathogen interaction,and plant hormone signal transduction.These annotations provide a valuable resource for investigating specific processes,functions and pathways during Brassia napus L.research.(3)By identifying differentially expressed genes,we screened a total of 639 specific unigenes fragments and analyzed for homology with rape genome,finally we got 53 EST sequences which has a complete ORF.Adopt the Brasicca napus L.drought resistant strain R29 as a template,28 related genes cloned.We also constructed an expression library driven by CaMV 35S(cauliflower mosaic virus 35S)promoter through Gateway system.(4)We have completed the transformation of Col-0 ecotypes Arabidopsis(more than 500)by Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method,and harvested 20,000 T0 generation seeds.We have identified 235 T1 positive plants by antibiotic screen and obtained seeds from 215 individuals.After breeding a generation,we screened by simulating stress conditions,obtain a mutant plant stress resistance phenotypes at Mannitol,NaCl and ABA plate were 163,384 and 366.Singled out the obvious phenotype plants sequencing,these plants are sequenced positive plants.By sequence alignment,we got 20 genes related to stress.Screened out the 20 genes of monoclonal from Agrobacterium library and transformed rape,subsequent to verify its functionality.