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作物学报 ›› 2023, Vol. 49 ›› Issue (1): 105-118.doi: 10.3724/SP.J.1006.2023.24004

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

应用RNA重测序分析低硫条件下大豆基因表达谱

王慧(), 吴志医, 张玉娥, 喻德跃()   

  1. 南京农业大学 / 国家大豆改良中心 / 作物遗传与种质创新国家重点实验室 / 农业农村部大豆生物学与遗传育种重点实验室, 江苏南京 210095
  • 收稿日期:2022-01-04 接受日期:2022-03-25 出版日期:2023-01-12 网络出版日期:2022-04-20
  • 通讯作者: 喻德跃
  • 作者简介:E-mail: wanghui0@njau.edu.cn
  • 基金资助:
    国家自然科学基金项目(32072080)

Transcriptional expression profiling of soybean genes under sulfur-starved conditions by RNA-seq

WANG Hui(), WU Zhi-Yi, ZHANG Yu-E, YU De-Yue()   

  1. National Center for Soybean Improvement / National Key Laboratory of Crop Genetics and Germplasm Enhancement / Key Laboratory of Biology and Genetic Improvement of Soybean, Ministry of Agriculture and Rural Affairs / Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2022-01-04 Accepted:2022-03-25 Published:2023-01-12 Published online:2022-04-20
  • Contact: YU De-Yue
  • Supported by:
    National Natural Science Foundation of China(32072080)

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摘要：

大豆是重要的粮油兼用作物, 其硫利用的研究不足。本研究评价了云梦六月花叶和沁阳大豆对低硫的耐性, 以这2个品种为材料, 利用RNA重测序技术分析了对照(+S)和缺硫(-S)水平下根和叶中的表达谱。结果表明, 云梦六月花叶对低硫表现为耐性, 沁阳大豆对低硫表现为敏感。表达谱分析在云梦六月花叶和沁阳大豆的叶中分别鉴定到9064个和9795个低硫响应的差异表达基因, 根中分别鉴定到3185个和5006个差异表达基因。KEGG富集分析发现, 2个材料叶中有9个共有途径, 仅植物MAPK信号途径富集更多的上调表达基因。2个材料根中有18个共有途径, 其中9个途径在2个材料的中对低硫的响应一致, 4个途径包含更多的上调表达基因, 5个途径包含更多的下调表达基因。在其余9个途径中, 云梦六月花叶包含更多的上调表达基因。大豆硫酸根转运蛋白基因对硫酸根的吸收和转运非常重要, 在表达谱中鉴定到27个硫酸根转运蛋白基因, 分属4个亚组, 亚组1、2、4的基因多受低硫诱导, 亚组3的基因对低硫的响应较为复杂。基于富集分析结果, 本研究从植物MAPK信号途径中克隆了一个受低硫诱导的基因GmEIL1, 通过转化大豆毛状根证明该基因参与大豆硫利用的调控。本研究结果为深入探索大豆硫利用效率的遗传机理奠定了基础, 为大豆耐低硫育种提供了候选基因。

关键词: 大豆, 低硫, RNA重测序, 硫酸根转运蛋白, EIN3/EIL

Abstract:

Soybean is an important grain and oil crop. Few researches focused on soybean sulfur utilization. Here, the tolerance of Yunmengliuyuehuaye (YM) and Qinyangdadou (QY) to low sulfur were evaluated. The gene expression profiles of roots and leaves of the two materials under the control (+S) and sulfur deficiency (-S) environments were analyzed via RNA resequencing. The results showed that YM was tolerance to low sulfur and QY was susceptibility to low sulfur. 9064 and 9795 differentially expressed genes (DEGs) were identified in leaves of YM and QY, and 3185 and 5006 DEGs were identified in roots of YM and QY, respectively. KEGG enrichment revealed that nine pathways were common in the two material leaves, of which MAPK signaling pathway—plant specially enriched more upregulated expressed genes. There were 18 common pathways in both roots. Nine of them responded consistently to low sulfur in YM and QY, of which four contained more up-regulated genes and five contained more downregulated genes. In the remaining nine pathways, YM contained more upregulated expressed genes. Soybean sulfate transporter genes were important for the absorption and transportation of sulfate. In the transcriptome, 27 soybean sulfate transporter genes were identified. These genes belonged to 4 subgroups respectively. Most of the genes in subgroups 1, 2, and 4 were induced by low sulfur, and the genes in subgroup 3 responded to low sulfur in a complex way. KEGG showed that the upregulated gene of GmEIL1 (ethylene-insensitive 3-like) in the MAPK signaling pathway-plant was clone. The gene was involved in the regulation of soybean sulfur utilization in the soybean chimeras with transgenic hairy root. These results provide a basis for deeply exploring the genetic mechanism of soybean sulfur utilization efficiency and candidate genes for soybean tolerance to low sulfate breeding in the future.

Key words: soybean, low sulfur, RNA-seq, sulfate transporter, EIN3/EIL

表1

引物序列"

基因名称
Gene name		 上游引物
Forward primer (5°-3°)		 下游引物
Reverse primer (5°-3°)		 用途
Application
GmEIL1 TTCAGCAGCCTTCTTTCCCAA AGCAAAACCATGGCGAGTTTC 定量PCR
qRT-PCR
GmTubulin GGAGTTCACAGAGGCAGAG CACTTACGCATCACATAGC 内参基因
Reference gene
EFR1 (Glyma.13G123100) TCCAAAGCCACTAACAAAAA CTCAGAACCCAAGTCCTCCA 定量PCR
qRT-PCR
GmEIL1 ACCACGGTGACCCATTTCTGTATCT TGATAATGATGATGAGGTTTCCAGC 基因克隆
Gene cloning
OE-GmEIL1 CAGGTCGACTCTAGAGGATCCGCCACCATGATGATGATGCTTGAAGA GGGAAATTCGAGCTCGGTACCTCACTGATACCAAATAGAGA 构建过表达载体
Construction of
overexpression vectors
RI-GmEIL1 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCCTTGAAGATATGGGATTTTG GGGGACCACTTTGTACAAGAAAGCTGGGTGAACCGAACTTTATCCTTCCA 构建干扰载体
Construction of RNAi vectors

图1

云梦六月花叶和沁阳大豆的低硫表型鉴定 +S和-S分别表示对照和缺硫处理。YM: 云梦六月花叶; QY: 沁阳大豆; *、**、***分别表示在0.05、0.01、0.001水平显著差异。"

附图1

21个差异表达基因的qRT-PCR与转录组重测序结果 log2(FC): YM和QY间差异表达基因改变倍数的对数(log2)转换值。L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

图2

云梦六月花叶和沁阳大豆的叶和根中的差异表达基因数 L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

图3

KEGG途径 L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

图4

共同富集途径中上调和下调差异表达基因的占比 富集在该途径的差异表达基因数列在括号内。L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

图5

硫酸根转运蛋白基因的表达谱 数值为对照和处理间差异表达基因改变倍数的对数(log2)转换值, 灰色空格表示没有显著差异。L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

图6

EILs蛋白序列的系统进化树(A)与大豆EIL基因的表达热图(B) 数字为对照和处理间差异表达基因改变倍数的对数(log2)转换值, 灰色空格表示没有显著差异。L和R分别表示叶和根。YM: 云梦六月花叶; QY: 沁阳大豆。"

附表1

2个大豆材料间差异表达的硫酸根转运蛋白基因"

基因名
Gene name		 R-YM/R-QY L-YM/L-QY
对照 Control 缺硫 Low sulfate 对照 Control 缺硫 Low sulfate
GmSULTR1;1a — — 2.93 —
GmSULTR2;1b -2.50 — 1.57 —
GmSULTR2;1c -2.15 — 2.66 —
GmSULTR2;2c -1.38 — — —
GmSULTR3;1a — -1.45 1.38 —
GmSULTR3;2a -1.48 -1.97 1.86 —
GmSULTR3;3b -3.09 -2.25 — —
GmSULTR3;4b — 2.63 4.80 —
GmSULTR3;4c — — — 1.41
GmSULTR3;5b 6.17 — 6.92 —
GmSULTR4;1 -1.05 — — —
GmSULTR4;2 — — 1.31 —

图7

GmNIL1的组织表达"

图8

大豆转基因嵌合体中GmEIL1基因表达量(A)、地上部和毛根中硫酸根含量(B和C)和ERF1 (Glyma.13G123100)基因的表达量(D) +S: 对照; -S: 缺硫。OE-EV、OE-EIL1、RI-EV、RI-EIL1分别表示转过表达空载、GmEIL1基因过表达、干扰空载、GmEIL1基因干扰的转基因嵌合体。*、**、***分别表示在0.05、0.01、0.001水平显著差异。"

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