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Transcriptomic Study of the Salt-Stress Response in the Rare Recretohalophyte Reaumuria Trigyna Native to Alxa Desert

Author: DangZhenHua
Tutor: WangYingChun
School: Inner Mongolia University
Course: Botany
Keywords: Reaumuria trigyna Recretohalophyte Tanscriptome Illuminasequencing Salt-stress response DDRT-PCR SSR loci identify
CLC: Q943
Type: PhD thesis
Year: 2013
Downloads: 92
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Abstract


Reaumuria trigyna is an endangered small shrub endemic to the Eastern Alxa-Western Ordos area in Inner Mongolia. This dicotyledonous recretohalophyte has developed unique morphological characteristics and adaption stratigies that allow it to tolerate the stress imposed by semi-desert saline soil. The remarkable capabilities to tolerance salty soil make it excellent candidates to investigate salt-tolerance mechanisms and to identify effective salt-response genes in the plant. However, it is impossible to explore the molecular mechanisms underlying this tolerance without detailed genomic information. At the present study, two sequencing libraries prepared from control (C21) and NaCl-treated samples (T43) were sequenced using short reads sequencing technology (Illumina) to obtain the transcriptome information of R. trigyna, and to investigate changes in the transcriptome of the species in response to salt stress. Based on the RNA-seq data, possible roles of the ion homeostasis regulation and the reactive oxygen species (ROS) scanvenging system played in salt stressed R. trigyna were discussed. In addition, DDRT-PCR technique was attemped to rapidly identify salt-stress induced genes from the massive amount of transcriptome sequencing data. Simple sequence repeat (SSR) locis were mined and validated. The results of the present study are as follow:1. In total,26.51and28.17million raw reads were generated from C21and T43libraries, respectively. The overall sequencing outputs were over than5Gigabase, and the transcriptome data platform was constructed. Among all the raw reads,more than92%had Phred-like quality scores at the Q20level (an error probability of1%). These were used for de novo assembly.2.68076(C21) and71194(T43) Unigenes were assembled from the Clean reads of two transcriptomes. After removal of redundancy,65340All-Unigene were clusted by combining C21-and T43-Unigenes. Among65340Unigenes,35495(52.27%) showed significant BLAST hits in the public databases.13552Unigenes were categorized into44Gene Ontology (GO) terms,10968Unigenes were subjected to25Clusters of Orthologous Groups (COG) families, and35271Unigenes were mapped to119canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Among the30randomly selected Unigenes,26Unigenes yielded expected fragments and the other four failed to produce amplification products, yielding a success rate of86.7%.3. Digital gene expression annotation and expression pattern of the differentially expressed genes showed that, among A11-Unigenes,32697were up-regulated and31997were down-regulated upon treatment of R. trigyna seedlings with salt solution.5032significantly differentialy expressed genes (DEGs) were identified, including2370up-and2662down-regulated DEGs.1947DEGs were enriched in27GO terms, and2086DEGs were enriched in33metabolic pathways. Among the Unigenes,135, 72, and25genes were identified to be related to regulation of K+uptake, H+pumping, and Na+efflux, and298Unigenes were predicted to encode antioxidative enzymes related to ROS scavenging. According to the gene expression patterns, the possible role played of the ion homeostasis regulation and the ROS scavenging system in response to salt stress of R. trigyna were discussed. The expression patterns of30randomly selected genes resulted from quantitative reverse transcription PCR (qRT-PCR) were basically consistent with their transcript abundance changes identified by RNA-seq.4. Rapidly identified the salt-stress induced genes from the massive amount of transcriptome sequencing data by DDRT-PCR combined with local BLAST analysis. The expression patterns of the18genes identified by DDRT-PCR detected by qRT-PCR were highly consistent with the digital gene expression data for the transcriptome database.5.225SSR motifs and6215SSRs were searched in65340Unigenes with the occurrence frequency of10%. Among Unigenes containing SSR locis,4153were successfully designed with the PCR primers, with the uccessfully rate of66.82%. PCR experiments showed that217of220randomly selected primer pairs could be yealied expected PCR products.

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