Summary: Popular rice mega varieties lack sufficient key micronutrients (e.g., Fe, Zn), vitamins and a balanced amino acid composition that are essential for a healthy diet. The major bottleneck for improving the nutritional quality of popular rice varieties through conventional breeding or gene technology is our lack of an integrated understanding of the biochemical and molecular processes that occur during rice grain filling (and their determining genes or loci). In this project, we will perform molecular expression profiling on specific tissue layers of the rice grain. To perform this experiment, the material will be developing rice seeds from plants grown hydroponically under controlled greenhouse conditions. Then, the laser microdissection approach will be applied to dissect different parts of the grain (i.e, vascular trace, aleurone, nucellar epidermis, etc). Total RNA will be extracted from these dissected parts and RNA sequencing will be performed. In this project, we will learn how the synthesis and deposition of grain nutrients is regulated, particularly, during grain filling.
Overall Design: 47 samples were collected and 39 samples passed QC. Biological replicates of poor quality (i.e., with low correlation coefficients) were excluded, therefore, 28 samples in total were used for detailed analysis [NE_4DAF(3), OVT_4DAF(3), CC_4DAF(2), EN_4DAF(1), NE_8DAF(3), OVT_8DAF(2), CC_8DAF(1), EN_8DAF(2), NE_16DAF(3), OVT_16DAF(1), CC_16DAF(2), EN_16DAF(2), AL_16DAF(3)]
Rice plants (Oryza sativa cv. Nipponbare) were grown in hydroponic solutions at 28°C. Solutions for the hydroponic system were prepared as previously described (Wang et al. 2013).
Treatment Protocol:
Rice grains were collected at 04, 08 and 16 days after flowering (DAF), de-husked and immediately frozen by placing the collection tubes in liquid nitrogen. Five sub-regions of the rice grains, including CC, NE, OVT, EN and AL were dissected for further analysis (see below). The time points were selected based on the reported differentiation of the main cell types (Krishnan and Dayanandan 2003). NE is differentiated into a single layer cell at around 4 DAF, while the enlarged cells and thickenings of NE are noticed at 8 DAF (Krishnan and Dayanandan 2003). CC are visible as greenish cell layer from 4 DAF to 16 DAF. OVT locates in the ventral side of the endosperm, in conjunction with NE (Krishnan and Dayanandan 2003). EN cells are gradually developing and enlarging through 4 to 16 DAF, and the outer layer of EN cells differentiate into AL at about 16 DAF.
Extract Protocol:
The RNA extraction was done with the Qiagen RNeasy Micro Kit following manufacturer’s instruction manual. All RNA samples were adjusted to a concentration of 100 pg/µl prior to pre-amplification. Pre-amplification for the Illumina sequencing was done using the Clonetech SMARTer Ultra Low kit following the user’s manual with small modifications. In brief, the RNA samples were subjected to first strand cDNA synthesis, followed by 15 cycles of Long Distance (LD) PCR. The PCR amplified cDNA was subsequently purified by immobilizaiton on AMPure XP beads for 15 min at RT, resuspended in 15 µl nuclease-free water and stored at -20°C for further experiments and library preparation.
Library Construction Protocol:
The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing were performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).
