中国农大朱鸿亮团队利用CRISPR/Cas9编辑lncRNA1459并发现其调控番茄成熟过程

近日，中国农大朱鸿亮团队在Plant Journal 发表题为“CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters tomato fruit ripening'的研究论文。利用CRISPR/Cas9基因编辑技术敲除了lncRNA1459，并发现其延迟了番茄果实的成熟。

随着高通量测序技术的发展，越来越多的长非编码RNA被发现具有重要的生物学功能。然而大多数的lncRNA功能未知。该研究团队以前发现了一个番茄成熟相关的lncRNA：lncRNA1459。本研究中，该团队获得了lncRNA1459的全长序列，研究发现lncRNA1459有两种转录本。除此之外，还发现lncRNA1459特异的定位于细胞核中。为了深入研究lncRNA1459，研究人员利用CRISPR/Cas9技术对lncRNA1459进行了基因编辑，获得了lncRNA1459的功能缺失突变体。相比于野生型，番茄lncRNA1459的功能缺失突变体的成熟进程受到了显著的抑制。lncRNA1459的功能缺失突变体的乙烯产生和番茄红素的积累也受到了显著的抑制。乙烯和类胡罗卜素合成相关的基因的表达在lncRNA1459的功能缺失突变体中也受到了显著的下调。lncRNA1459敲除突变体中许多与果实成熟相关的基因也发生了明显的变化。该研究对于理解lncRNA1459在果实成熟中的作用具有重要意义。

原文链接

http://onlinelibrary.wiley.com/doi/10.1111/tpj.13872/abstract

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Zhu B, Yang Y, Li R, Fu D, Wen L, Luo Y*, Zhu H* (2015) RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening. Journal of Experiment Botany 66: 4483-4495

Figure 2. Loss-of-function of lncRNA1459 specifically delayed tomato fruit ripening in T1.

(a) One target site was designed on lncRNA1459 for CRISPR/Cas9. Target site was located on blue box whose sequence were detected from 3’ RACE fragment sequences of lncRNA1459. Furthermore, red boxes showed the 5’ RACE fragment sequences of lncRNA1459 and black boxes showed the RNA-seq data.

(b) Expression level of lncRNA1459 in WT and CR-lncRNA1459 mutants, determined by qPCR. lncRNA1459 expression level were normalized by Actin expression level. The relative lncRNA1459 expression level in WT was assigned a value of 1. Error bars indicate ±SD of three biological replicates, with each measured in triplicate. Samples marked with various letters show a significant difference at p<>

(c) Phenotype of independent homozygous mutants CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 in T1 generation. CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 fruits were compared with WT fruits at 30 DPA, 35DPA, 40DPA, 45DPA and 50 DPA ripening stages, showing different color from 35DPA.

(d) Genotype of CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 in T1 generation. Both CR-lncRNA1459-8-3 and CR-lncRNA1459-21-1 were homozygous mutation. Black dashed lines indicated base pair deletion. Red letters were target site sequences. Letters with underline were PAM.