番茄 MADS 盒转录因子 RIN 的全基因组鉴定及其长非编码 RNA 靶基因的功能分析。
Genome-wide identification of long non-coding RNA targets of the tomato MADS box transcription factor RIN and function analysis.
机构信息
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
EG12 Science Centre School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China.
出版信息
Ann Bot. 2019 Feb 15;123(3):469-482. doi: 10.1093/aob/mcy178.
BACKGROUND AND AIMS
In recent years, increasing numbers of long non-coding RNAs (lncRNAs) have been identified in humans, animals and plants, and several of them have been shown to play important roles in diverse biological processes. However, little work has been performed on the regulation mechanism of lncRNA biogenesis and expression, especially in plants. Compared with studies of tomato MADS-box transcription factor RIPENING INHIBITOR (RIN) target coding genes, there are few reports on its relationship to non-coding RNAs. The aim of the present study was to identify and explore the specific role of RIN target lncRNAs in tomato fruit development and ripening.
METHODS
lncRNA targets of RIN were identified by chromatin immunoprecipitation sequencing (ChIP-seq) combined with RNA deep sequencing analysis. Six selected lncRNA targets were validated by quantitative real-time PCR, ChIP and electrophoretic mobility shift assays, and we further confirmed differential expression between wild-type and ripening-deficient mutant fruit, and RIN direct binding in the promoter regions. By means of virus-induced gene silencing (VIGS) assays and a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing strategy, the ripening-related function of a specific target lncRNA (lncRNA2155) was studied.
KEY RESULTS
We identified 187 lncRNAs as direct RIN targets, which exhibited RIN binding sites in their promoters and showed different expression between the wild-type and rin mutant. Six target lncRNAs were shown to bind with RIN directly in their promoters in vivo and in vitro. Moreover, using CRISPR/Cas9 technology to knock out the locus of the target lncRNA2155 indicated that it delayed fruit ripening in tomato.
CONCLUSIONS
Collectively, these findings provide new insight into RIN in the transcriptional regulation of lncRNAs and suggest that lncRNAs will contribute to a better understanding of the RIN regulatory network that controls fruit ripening.
背景与目的
近年来，人类、动物和植物中越来越多的长链非编码 RNA（lncRNA）被鉴定出来，其中一些已被证明在多种生物过程中发挥着重要作用。然而，关于 lncRNA 生物发生和表达的调控机制的研究很少，特别是在植物中。与番茄 MADS-box 转录因子 RIPENING INHIBITOR（RIN）靶编码基因的研究相比，关于其与非编码 RNA 的关系的报道较少。本研究的目的是鉴定和探索 RIN 靶标 lncRNA 在番茄果实发育和成熟中的特定作用。
方法
通过染色质免疫沉淀测序（ChIP-seq）结合 RNA 深度测序分析鉴定 RIN 的 lncRNA 靶标。通过定量实时 PCR、ChIP 和电泳迁移率变动分析验证了 6 个选定的 lncRNA 靶标，并进一步证实了野生型和成熟缺陷突变体果实之间的差异表达，以及 RIN 在启动子区域的直接结合。通过病毒诱导基因沉默（VIGS）试验和簇状规则间隔短回文重复（CRISPR）/CRISPR 相关蛋白 9（Cas9）基因组编辑策略，研究了特定靶标 lncRNA（lncRNA2155）的成熟相关功能。
主要结果
我们鉴定了 187 个 lncRNA 作为 RIN 的直接靶标，它们在启动子中具有 RIN 结合位点，并在野生型和 rin 突变体之间表现出不同的表达。在体内和体外均证明了 6 个靶标 lncRNA 直接与 RIN 在其启动子上结合。此外，利用 CRISPR/Cas9 技术敲除靶标 lncRNA2155 的基因座表明，它延迟了番茄果实的成熟。
结论
总之，这些发现为 RIN 在 lncRNA 的转录调控中的作用提供了新的见解，并表明 lncRNA 将有助于更好地理解控制果实成熟的 RIN 调控网络。
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