IC4R014-RNA-Seq-2013-23322175

Project Title

• Transcriptomic analysis of rice (Oryza sativa) endosperm using the RNA-Seq technique

The Background of This Project

• The endosperm plays an important role in seed formation and germination, especially in rice (Oryza sativa). In this project, the researchers used a high-throughput sequencing technique (RNA-Seq) to reveal the molecular mechanisms involved in rice endosperm development.

Plant Culture & Treatment

• The endosperm were derived from rice (O. sativa L.ssp. indica cv.9311), which were grown in a greenhouse at Wuhan University, China. Caryopses were tagged at the initiation of pollination. The samples consisted of three replicates arranged in a randomized complete block design. For each replicate, development seed were collected from 15 different plants at 3, 6, and 10 DAP during the growing season. The three-stage endosperm was taken from caryopses with microdissection needles under a dissection microscope (Olympus, Tokyo, Japan). The materials were immediately frozen in liquid nitrogen and kept at -80℃ until required (for RNA extractions).

Illumina Sequencing

• Total RNA was prepared using Trizol according to the manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). The yield and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm and the integrity of all RNA samples was monitored on 1 % agarose gels. The total RNA samples were pooled into three-stage samples and 10 lg of total RNA from each sample was used to isolate poly (A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNASeq 8 Sample Prep Kit (Illumina). Each library had an insert size of 200 bp, and 42–50 bp sequences were generated via Illumina HiSeqTM 2000.

Research Findings

• A large number of genes related to ribosomes, the spliceosome and oxidative phosphorylation were found to be expressed in the early and middle stages of rice endosperm development. Plant hormone, galactose metabolism and carbon fixation related genes showed a significant increase in expression at the middle stage, whereas genes for defense against disease or response to stress as well as genes for starch/sucrose metabolism were strongly expressed during the later stages of endosperm development.

• By comparison with the rice genome and trimming, all 35.9 high-quality million clean reads were assembled into 23,836 genes. Among the three stages, the number of genes found were 21,596 (R1), 20,910 (R2) and 19,459 (R3), providing massive data for endosperm development analysis. From these numbers, there is an apparent decline in quantity during endosperm development. A Venn diagram shows the overlapping relationship between the three stages (Fig. 1).

Figure 1. Venn diagram showing the genes expressed in each of the three stages of rice endosperm development.

• From the analysis of differentially expressed genes (DEG) between samples ,the researchers could also identify the change in gene expression between three stages (Table 1). They identified 10,371 DEGs in three comparisons (R1-v-R2, R2-v-R3 and R1-v-R3). The overall trend in all the comparisons was downregulation of gene expression. Only a minority of genes were up-regulated during rice endosperm development.

Table 1. All 10,371 DEGs divided according to the folds of RPKM value between two stages in rice endosperm development

• To identify clusters with functional enrichment, the researchers performed hierarchical clustering of the expression patterns of all DEGs and found that there was a similarity relationship in gene expression among three stages (Fig. 2). The hierarchical clustering analysis indicates that the majority of genes were downregulated in all three comparisons, and that a minority of genes were up-regulated.

Figure 2. Hierarchical cluster analysis of gene expression based on log ratio RPKM data.

• Through the GO analysis, all genes were separated into three major parts (i.e., biological process, molecular function, and cellular component). Using BLAST2GO (version 2.3.5), a total of 157,650 GO terms were associated with all 23,836 genes and classified into 41 functional subcategory annotations (Fig. 3).The GO annotation assignment determined that the genes expressed in this study encode diverse structural, regulatory and metabolic proteins.

Figure 3. GO classifications of genes.The results are summarized in three main categories: biological process, molecular function and cellular component by GO analysis. a GO classifications of all genes between three stages. b GO classifications of all 10,371 DEGs among three contrasts. c GO analysis of the up-regulated and down-regulated genes in R1-v-R2. d GO analysis of the up-regulated and downregulated genes in R2-v-R3 (*P\0.05)

• To reveal the molecular mechanisms of events involving TFs in rice endosperm development, the researchers used the Rice TF database to search for TFs. In total, 1,118 putative TF genes of 55 TF families were identified in 23,836 genes from RNA-Seq analysis. This provided a deeper understanding of the role of TFs during rice endosperm development.

Labs working on this Project

• State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, China

Corresponding Author

• jbwang@whu.edu.cn