IC4R020-RNA-Seq-2016-26752408

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Project Title

  • Transcriptome Analysis of Salt Stress Responsiveness in the Seedlings of Dongxiang Wild Rice (Oryza rufipogon Griff.)


The Background of This Project

  • Rice (Oryza sativa L.) is one of the most important food crops in the world and also a model for genomic research in monocots. However, salinity is one of the most devastating abiotic stresses in rice, and the salt-affected soils currently account for about 20% of the total paddy rice planting area.More seriously, the area of salt-affected irrigated land is expanding and spreading in China.
  • Dongxiang wild rice (Oryza rufipogon Griff., hereafter referred as DXWR) is the progenitor of cultivated rice (Oryza sativa L.). DXWR, a Chinese type of wild rice grown in Jiangxi Province (28°14’N latitude and 116°30’E longtitude), is considered to be the northernmost region in the world where O. rufipogon is found. DXWR grows in the natural habitats and possesses various characteristics resistant to biotic and abiotic stresses and abundant genetic diversity which have been lost in the cultivated rice. Thus, it is an extremely important resource for providing a valuable gene pool for rice genetic improvement.
  • Recently, next-generation high-throughput RNA sequencing technology (RNA-Seq) could overcome the drawbacks of array-based technologies. With the high resolution and sensitivity, the RNA-Seq could be used for discovering novel splice junctions, novel transcripts, alternative transcription start sites and rare transcripts. Moreover, RNA-Seq data revealed a high level of reproducibility in both technical and biological replicates. So far, the global gene expression in various plants has been profiled by RNA-Seq.
  • Core sets of transcription factor family genes are differentially expressed in response to elevated external salinity, including basic leucine zipper (bZIP), WRKY,APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), MYB, basic helix-loophelix (bHLH) [38], and NAC [39] families. These transcription factors, in turn, regulate the expression levels of various genes that may ultimately influence the level of salt tolerance of plants. In rice, recent studies have indicated that a large number of transcription factors were involved in salt stress response, such as OsMYB91, OsbZIP71, OsWRKY42,SERF1, OsTZF1, and OsNAC5.


Plant Culture & Treatment

  • Seeds of Dongxiang wild rice (Oryza rufipogon Griff.; Dongxiang County, Jiangxi Province) and rice Xieqingzao B (O. sativa L. ssp. indica) were immersed in distilled water in the dark,and the uniformly germinated seeds were sown in 96-well plates supported by a plastic container. Seeds were grown in a growth chamber, as previously described [12]. The growth culture solution was renewed every 3 days. After the seedlings had been grown for 14 days, they were transferred on their 96-well plates into containers filled with 200 mM NaCl solution, or with control solution for 12 days. The seedlings were then recovered under normal solution for 3 days, and survival rates were calculated. The experiment was a randomized complete block design with three replications. For RNA-Seq analysis, 14-day-old seedlings of Dongxiang wild rice were grown with or without 200 mM NaCl treatment for 3 days and then the leaves (penultimate leaves) and total roots (separated from the culture solution and washed carefully) of these seedlings were collected and immediately frozen in liquid nitrogen, respectively. For RNA extraction from each treatment group, 10 plants were collected and mixed, to minimize the effect of transcriptome unevenness among plants.


Illumina sequencing

  • the Illumina HiSeq 2000 platform was used to analyze the transcriptome profiles of the leaves and roots at the seedling stage under salt stress compared with those under normal conditions.


Research Findings

  • To investigate the character of salt stress resistance in the DXWR seedlings, two-week-old seedlings of DXWR and Xieqingzao B (O. sativa L. ssp. indica, hereafter referred as XB), which is a representative maintainer line in hybrid rice breeding system in China [12] were exposed to the hydroponic solution with 200 mM NaCl for 12 days. When grown under normal, the DXWR and XB seedlings were all green and alive. After exposure to the salt stress, most DXWR seedlings remained green and showed continuous growth, whereas XB seedlings were all dead (Fig 1).


'Fig 1. The seedlings of DXWR showed stronger salt resistance than the seedlings of XB.'


  • A total of 15,636 and 17,937 transcripts were assigned GO terms for the DEGs in the LS vs. LCK and RS vs. RCK, respectively. Among the 15,636 transcripts from the LS vs. LCK (Fig 3A), there were 5,633 transcripts at the cellular level, 5,068 transcripts at the molecular level and 4,935 transcripts at the biological level. Among the 17,937 transcripts from the RS vs. RCK (Fig 3B), there were 6,302 transcripts at the cellular level, 5,907 transcripts at the molecular level and 5,728 transcripts at the biological level.


IC4R020-RNA-Seq-2016-26752408-f3a.png
'Fig 3. Gene ontology (GO) classification of the unigenes from the LS-vs-LCK (A) and RS-vs-RCK (B).'


  • The researcher further identified over-represented KEGG Orthology (KO) terms (Q-value < 0.05), and classified them into 10 categories, respectively (Fig 4). As shown in Fig 4, these transcripts belonged mainly to the following KEGG pathways both in the LS vs. LCK and RS vs. RCK: Biosynthesis of other secondary metabolites, carbohydrate metabolism, global map, metabolism of terpenoids and polyketides, translation, and transport and catabolism.


IC4R020-RNA-Seq-2016-26752408-f4a.png
'KEGG pathway assignments in the LS vs. LCK (A) and RS vs. RCK (B). The represented categories (Q-value� 0.05) and the number of transcripts predicted to belong to each category are shown.'


Labs working on this Project

  • College of Life Sciences, Jiangxi Normal University, Nanchang, China
  • Institute for Advanced Study,Jiangxi Normal University, Nanchang, China


Corresponding Author

  • Yi Zhou:zhouyi25@mail2.sysu.edu.cn; & Jiankun Xie: xiejiankun@yahoo.com