全球鉴定和分析揭示了与同源四倍体水稻减数分裂和低育性相关的差异表达 lncRNAs。
Global identification and analysis revealed differentially expressed lncRNAs associated with meiosis and low fertility in autotetraploid rice.
机构信息
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, 510642, China.
Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University, Guangzhou, 510642, China.
出版信息
BMC Plant Biol. 2020 Feb 19;20(1):82. doi: 10.1186/s12870-020-2290-0.
BACKGROUND
Autotetraploid rice is a useful germplasm for polyploid rice breeding. Our previous research showed that non-coding RNAs might be associated with low fertility in autotetraploid rice. However, little information is available on long non-coding RNAs (lncRNAs) involved in the low fertility of autotetraploid rice. In the present study, RNA-seq was employed to detect the differentially expressed meiosis-related lncRNAs in autotetraploid rice, and gene overexpression and knock out experiments were used to validate the potential function of candidate lncRNA.
RESULTS
A total of 444 differentially expressed lncRNAs (DEL) were detected during anther and ovary meiosis in autotetraploid rice. Of these, 328 DEL were associated with the transposable elements, which displayed low expression levels during meiosis in autotetraploid rice. We used rapid amplification of cDNA ends (RACE) assay to validate 10 DEL and found that the lncRNAs were not assembly artifacts, and six of them were conserved in tetraploid rice. Moreover, 237 and 20 lncRNAs were associated with pollen mother cell (PMC) and embryo sac mother cell (EMC) meiosis in autotetraploid rice, respectively. The differential expressions of some meiosis-related targets and its DEL regulator, including MEL1 regulated by TCONS_00068868, LOC_Os12g41350 (meiotic asynaptic mutant 1) by TCONS_00057811 in PMC, and LOC_Os12g39420 by TCONS_00144592 in EMC, were confirmed by qRT-PCR. TCONS_00057811, TCONS_00055980 and TCONS_00130461 showed anther specific expression patterns and were found to be highly expressed during meiosis. CRISPR/Cas9 editing of lncRNA57811 displayed similar morphology compared to wild type. The overexpression of lncRNA57811 resulted in low pollen fertility (29.70%) and seed setting (33%) in rice.
CONCLUSION
The differential expression levels of lncRNAs, associated with transposable elements and meiosis-regulated targets, might be endogenous noncoding regulators of pollen/embryo sac development that cause low fertility in autotetraploid rice. The results enhance our understanding about rice lncRNAs, and facilitate functional research in autotetraploid rice.
背景
同源四倍体水稻是一种用于多倍体水稻育种的有用种质。我们之前的研究表明，非编码 RNA 可能与同源四倍体水稻的低育性有关。然而，关于参与同源四倍体水稻低育性的长非编码 RNA（lncRNA）的信息很少。在本研究中，我们采用 RNA-seq 检测同源四倍体水稻减数分裂过程中差异表达的与减数分裂相关的 lncRNA，并通过基因过表达和敲除实验验证候选 lncRNA 的潜在功能。
结果
在同源四倍体水稻的花药和子房减数分裂过程中，共检测到 444 个差异表达的 lncRNA（DEL）。其中，328 个 DEL 与转座元件相关，在同源四倍体水稻减数分裂过程中表达水平较低。我们使用快速扩增 cDNA 末端（RACE）实验验证了 10 个 DEL，并发现这些 lncRNA 不是组装伪影，其中 6 个在四倍体水稻中保守。此外，在同源四倍体水稻中，237 个和 20 个 lncRNA 分别与花粉母细胞（PMC）和胚囊母细胞（EMC）减数分裂有关。一些减数分裂相关靶标及其 DEL 调节剂的差异表达，包括由 TCONS_00068868 调控的 MEL1、由 TCONS_00057811 调控的 LOC_Os12g41350（减数分裂不联会突变体 1），在 PMC 中，和由 TCONS_00144592 调控的 LOC_Os12g39420，在 EMC 中，通过 qRT-PCR 得到证实。TCONS_00057811、TCONS_00055980 和 TCONS_00130461 表现出花药特异性表达模式，并且在减数分裂过程中表达水平较高。lncRNA57811 的 CRISPR/Cas9 编辑显示出与野生型相似的形态。lncRNA57811 的过表达导致水稻花粉育性（29.70%）和结实率（33%）降低。
结论
与转座元件和减数分裂调控靶标相关的 lncRNA 的差异表达水平可能是同源四倍体水稻花粉/胚囊发育低育性的内源性非编码调控因子。这些结果提高了我们对水稻 lncRNA 的认识，并促进了同源四倍体水稻的功能研究。
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