概要: Purpose: Comprehensive comparison of gene-expression profiles between 3mlpa (mutant with low phytic acid) and 3MWT (non-mutant with normal phyic acid) soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. Methods: mRNA sequencing reads (101SE) were generated in triplicate from five seed developmental stages of 3mlpa and 3MWT soybean line using Illumina HiSeq 2000. Results: A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. Conclusion: This study provides a global perspective of transcriptomal changes during soybean seed development in 3mlpa mutant. The results suggest that low phtic acid-causing mutations in 3mlpa play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively.
项目整体设计: RNA-Seq of five seed developing soybean seeds from mips1/mrp-l/mrp-n triple mutant line (with low phytic acid) and MIPS1/MRP-L/MRP-N non-mutant line (with normal phytic acid). The mRNA libraries from three biological replicates of each sample were sequenced as 101 SE using Illumina HiSeq 2000.
For each of the two experimental lines, 48 plants were grown in 12 pots (four plants per pot) containing Metro-Mix® 360 (Sun Gro) soilless media, over-layered with topsoil GardenPro ULTRALITE. All plants were grown in the same growth chamber unit, with controlled conditions, as follows: 14/10 h (day/night) photoperiod, 24/16 °C (day/night), light intensities in the range of 300 and 400 μE and 50–60 % relative humidity. About 41–47 plants from each experimental line were used for sampling developing seeds.
处理方案:
Seed length was the criterion for sampling different developmental stages. Pods were randomly selected, opened and, seed length was measured. Five developmental stages were defined by seed length as: (Stage 1) between 2-4 mm; (Stage 2) between 4-6 mm; (Stage 3) between 6-8 mm; (Stage 4) between 8-10 mm; and, (Stage 5) between 10-12 mm. Three biological replicates for each stage were taken, where each replicate sample was represented by a minimum of 10-15 seeds (stages 1-2), and at least 3 seeds (stages 3-5), collected from different pods on separate plants. Sampled seeds were immediately frozen in liquid nitrogen and stored at -70 °C.
提取方案:
Whole soybean developing seeds sampled in triplicate from 3mlpa and 3MWT lines at five stages of seed development, were flash frozen, and total RNA was extracted using QIAGEN RNeasy Plant Mini Kit, with on column DNase digestion.
建库方案:
High quality total RNA samples were used to generate 30 mRNA libraries using standard Illumina TruSeq RNA sample preparation kit protocol.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
SINGLE
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2000
链特异性:
Specific
样本
基本信息:
样本描述:
生物条件:
实验变量:
方案:
测序信息:
质量评估:
数据来源
GEN样本编号
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物种
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组织
细胞类型
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疾病
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突变/变异
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Condition Detail
生长方案
处理方案
提取方案
建库方案
分子类型
库的片段类型
链特异性
库的链类型
加标(Spike-In)
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平均测序片段长度_1
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唯一比对率
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覆盖度
文章
Genome-wide transcriptome analyses of developing seeds from low and normal phytic acid soybean lines.