20171014植物界文献更新：番茄纳米孔测序；植物Cas9优化；水稻BR发现新蛋白；下胚轴伸长机制；两小花穗增产

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1Plant Cell：纳米孔测序技术从头组装番茄基因组

 De novo Assembly of a New Solanum pennellii Accession Using Nanopore Sequencing

First author:Maximilian HW Schmidt; Affiliations: RWTH Aachen University( 亚琛工业大学): Aachen, Germany Corresponding author: Bjoern Usadel

   Abstract：

    Updates in nanopore technology  have made it possible to obtain gigabases of sequence data. Prior to  this, nanopore sequencing technology was mainly used to analyze microbial samples. Here, we describe the generation of a comprehensive nanopore sequencing dataset with a median read length of 11,979 bp for a self-compatible accession of the wild tomato species Solanum pennellii. We describe the assembly of its genome to a contig N50 of 2.5 MB. The assembly pipeline comprised initial read correction with Canu and assembly with SMARTdenovo. The resulting raw nanopore-based de novo genome is structurally highly similar to that of the reference S. pennellii LA716 accession but has a high error rate and was rich in homopolymer deletions. After polishing the assembly with Illumina reads, we obtained an error rate of <0.02% when assessed versus the same Illumina data. We obtained a gene completeness of 96.53%, slightly surpassing (优于) that of the reference S. pennellii. Taken together our data indicate that such long read sequencing data can be used to affordably sequence and assemble gigabase-sized plant genomes.

    纳米孔技术的更新发展使得获得千兆碱基的测序数据成为可能。在此之前，纳米孔测序技术主要用于微生物样品的测序。本文利用纳米孔测序技术对已有报道基因组的野生番茄Solanum pennellii进行了测序，平均读长为11,979 bp。利用Canu进行原始reads纠错，然后利用SMARTdenovo进行从头组装，最终获得的组装contig N50长2.5 MB。利用纳米孔测序技术最终获得的基因组与之前S. pennellii LA716参考基因组结构上差不多，但错误率较高，且均聚物缺失丰富。作者进一步利用Illumina测序reads对基因组进行矫正，最终的错误率小于0.02%。基因完整性评估值为96.53%，优于S. pennellii的参考基因组。综上，利用纳米孔技术测序获得的长reads可以用来测序、组装以获取千兆级别的植物基因组。

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2Genome Biology: 高彩霞研究组在高保真CRISPR-Cas9基因组编辑方法研究中取得新进展

    Perfectly Matched 20-nucleotide Guide RNA Sequences Enable Robust Genome Editing Using High-fidelity SpCas9 Nucleases.

Dingbo Zhang, Huawei Zhang, Tingdong, Kunling Chen, Jinlong Qiu and Caixia GaoGenome Biology

DOI:10.1186/s13059-017-1325-9 

来源构官方：

    基因组编辑是生命科学新兴的颠覆性技术,特别是基于CRISPR-Cas9系统的基因组编辑工具近几年迅猛发展,在医疗、农业等领域得到广泛应用.然而,Cas9脱靶现象是目前限制其发挥巨大潜力的最主要问题之一,提高该系统的特异性一直是基因组编辑方法研究的焦点. 通过蛋白质工程的方法,美国两个课题组前期分别对Cas9蛋白进行定向改造,获得了三种特异性显著提高的Cas9蛋白变体:eSpCas9(1.0)、eSpCas9(1.1) 和 SpCas9-HF1.

    中国科学院遗传与发育生物学研究所高彩霞研究组近期的研究发现,这三种高保真的SpCas9核酸酶的基因组编辑活性会严格受到sgRNA向导序列(guide sequence)长度的影响.将向导序列设为与靶位点精确匹配的20个碱基,是确保三种高保真SpCas9核酸酶的活性的重要前提.

    为此,高彩霞研究团队将水稻tRNAGlu序列融合到U3启动子和sgRNA之间,利用细胞內源的RNase P和RNase Z将未成熟的sgRNA中的向导序列加工成为与靶序列精确匹配的20个碱基,通过这一策略能够将eSpCas9(1.0)、eSpCas9(1.1)和SpCas9-HF1的活性保持在与野生型SpCas9相当的水平,并且还保持其特异性. 

图：Guide sequence 对SpCas9 及其变体活性的影响

    该研究成果于2017年10月11日在线发表于Genome Biology杂志上(DOI:10.1186/s13059-017-1325-9).高彩霞研究组硕士生张定波和副研究员张华伟为该论文的并列第一作者,该研究得到科技部,农业部,中科院以及国家自然基金委的资助. 

Abstract

High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA–sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering.

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3Front Plant Sci: BR通过调控OFP1，（DLT1互作蛋白）调控水稻株型及穗粒形态  (来源：中科院遗传发育所储成才研究组）

 Front Plant Sci. 2017 Sep 27;8:1698. doi: 10.3389/fpls.2017.01698. eCollection  2017.

Brassinosteroids Regulate OFP1, a DLT Interacting Protein, to Modulate Plant Architecture and Grain Morphology in Rice.

Xiao Y^1,2, Liu D^1,2, Zhang G^1,2, Tong H³, Chu C^1,2.

• 1State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

• 2University of Chinese Academy of Sciences, Beijing, China.

• 3National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.

Abstract

Brassinosteroids (BRs) regulate important agronomic traits in rice, including plant height, leaf angle, and grain size. However, the underlying mechanisms remain not fully understood. We previously showed that GSK2, the central negative regulator of BR signaling, targets DLT, the GRAS family protein, to regulate BR responses. Here, we identified Ovate Family Protein 1 (OFP1) as a DLT interacting protein. OFP1 was ubiquitously expressed and the protein was localized in both cytoplasm and nucleus. Overexpression of OFP1 led to enlarged leaf angles, reduced plant height, and altered grain shape, largely resembled DLT overexpression plants. Genetic analysis showed that the regulation of plant architecture by OFP1 depends on DLT function. In addition, we found OFP1 was greatly induced by BR treatment, and OsBZR1, the critical transcription factor of BR signaling, was physically associated with the OFP1 promoter. Moreover, we showed that gibberellin synthesis was greatly repressed in OFP1 overexpression plants, suggesting OFP1 participates in the inhibition of plant growth by high BR or elevated BR signaling. Furthermore, we revealed that OFP1 directly interacts with GSK2 kinase, and inhibition of the kinase activity significantly promotes OFP1 protein accumulation in plant. Taken together, we identified OFP1 as an additional regulator of BR responses and revealed how BRs promote OFP1 at both transcription and protein levels to modulate plant architecture and grain morphology in rice.

机器翻译（请参考）：

    油菜素内酯（BR）调节水稻的重要农艺性状，包括植物高度，叶角度和粒度。然而，潜在的机制尚未完全了解。我们以前表明，BR信号的中枢负调节因子GSK2靶向了DLT，GRAS家族蛋白，以调节BR反应。在这里，我们将卵磷脂蛋白1（OFP1）鉴定为DLT相互作用蛋白。 OFP1被普遍表达，蛋白质位于细胞质和细胞核中。 OFP1的过表达导致叶角增大，植物高度降低，谷粒形状改变，很大程度上类似于DLT过表达植物。遗传分析表明，OFP1对植物结构的调节取决于DLT功能。此外，我们发现OFP1被BR处理极大地诱导，并且BRB信号的关键转录因子OsBZR1与OFP1启动子物理相关。此外，我们表明，赤霉素合成在OFP1过表达植物中被大大抑制，表明OFP1参与高BR或升高的BR信号传导对植物生长的抑制。此外，我们发现OFP1与GSK2激酶直接相互作用，抑制激酶活性显着促进植物中的OFP1蛋白积累。总之，我们将OFP1鉴定为BR反应的额外调节因子，并揭示BR在转录和蛋白质水平上如何促进OFP1调节水稻的植物结构和谷粒形态。

4Front Plant Sci：ERECTA通过激活生长素合成调控拟南芥下胚轴细胞伸长

Front Plant Sci. 2017 Sep 27;8:1688. doi: 10.3389/fpls.2017.01688. eCollection  2017.

ERECTA Regulates Cell Elongation by Activating Auxin Biosynthesis in Arabidopsis thaliana.

Qu X¹, Zhao Z¹, Tian Z¹.

Author information

• 1School of Life Sciences, University of Science and Technology of China, Hefei, China.

Abstract

The ERECTA family genes, ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2), encode leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Knocking out these three genes can cause severe phenotypes, which indicates that they play significant roles in plant growth and development. However, the molecular mechanism within remains unclear. Here we show that the short hypocotyl phenotypes of er erl1 erl2 mutants are mainly due to the defects of cell elongation rather than the cell division. In contrast, in the ERECTA overexpression transgenic plants, the hypocotyl length is increased with elongated cells. Moreover, we show that the er erl1 erl2 triple mutant contains a low level of auxin, and the expression levels of the key auxin biosynthesis genes are significantly reduced. Consistent with this observation, increasing exogenous or endogenous auxin levels could partially rescue the cell elongation defects of the er erl1 erl2 triple mutant. Therefore, our results provide a molecular basis for auxin mediated ERECTA control of the hypocotyl length in Arabidopsis thaliana.

KEYWORDS: Arabidopsis; ERECTA; auxin; cell elongation; hypocotyl length

    ERECTA家族基因ERECTA（ER），ERECTA-LIKE1（ERL1）和ERECTA-LIKE2（ERL2）在拟南芥中编码富含亮氨酸的重复受体样激酶。敲除这三种基因可能导致严重的表型，这表明它们在植物生长发育中起重要作用。然而，分子机制仍不清楚。在这里我们显示，er erl1 erl2突变体的短的胚轴表型主要是由于细胞延伸的缺陷而不是细胞分裂。相比之下，在ERECTA过表达转基因植物中，下胚轴长度随细胞增加而增加。此外，我们显示，er erl1 erl2三重突变体含有低水平的生长素，并且主要生长素生物合成基因的表达水平显着降低。与此观察一致，增加外源或内源性生长素水平可以部分挽救三突表型。因此，我们的研究结果为生长素介导的ERECTA控制拟南芥下胚轴长度提供了分子基础。

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5Plant biotechnology journal: 水稻两小花穗形态具有潜在的水稻增长能力（文章来源：中国农科院水稻所钱前组）

 "Two-florets spikelet" as a novel resource has the potential to increase rice yield. Plant Biotechnol J. 2017 Oct 11. doi: 10.1111/pbi.12849.

Abstract

Yield in rice (Oryza sativa) is determined by three major components: panicle number per plant, grain weight, and grain/spikelet number per panicle (Zhou et al., 2015). Grain number per panicle is one of the main targets, and mainly results from the number of spikelets. Traditionally, rice breeders have focused on the improvement of spikelet number per panicle and rarely focused on the number of florets because a normal rice spikelet has one fertile floret and produces one seed.

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