研究发现控制水稻粒形和稻米品质的重要基因

近日，中国科学院遗传与发育生物学研究所傅向东研究员领导的团队从优质杂交水稻不育系泰丰A中成功分离并克隆了一个控制水稻粒形和提升稻米品质的重要基因GW7。这一基因能通过改变细胞分裂模式，让稻米变得更为细长，有效地减少垩白率和垩白面积，从而提高稻米在外观、口感等方面的品质。该研究还表明将GW7和GS3基因的优异等位变异聚合并应用到我国高产籼稻中，可明显提高稻米品质，同时还可提高产量。该项研究为水稻高产优质分子模块设计育种提供了具有重要应用价值的新基因，也为揭示水稻品质和产量协同遗传改良的分子奥秘提供了新线索。

论文链接:Wang S，et al..The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality.

水稻氮利用效率改良研究取得重大突破

植物主要以铵态氮和硝态氮为主要氮源，中国科学院遗传与发育生物学研究所储成才研究员领导的团队研究表明，籼稻品种利用硝酸盐的能力显著高于粳稻品种，该团队通过图位克隆技术从籼稻中克隆出高氮利用效率基因NRT1.1B，NRT1.1B编码一个硝酸盐转运蛋白，在籼、粳稻间只有一个氨基酸的差别，且籼稻与粳稻呈现出显著的分化，各种证据表明，籼稻型具有更高的硝酸盐吸收及转运活性。尤为重要的是，籼稻中的硝酸盐同化过程的关键基因也被显著上调，这种结果导致籼稻具有更高的氮肥利用能力，说明NRT1.1B在粳稻氮肥利用效率改良上具有巨大应用价值。

论文链接:Hu B，et al..Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies.

Nature Genetics，2015，47(7):834－838.doi:10.1038/ng.3337.Published online:08 June，2015.

Abstract:Asian cultivated rice(Oryza sativa L.)consists of two main subspecies，indica and japonica.Indica has higher nitrate-absorption activity than japonica，but the molecular mechanisms underlying that activity remain elusive.Here we show that variation in a nitratetransporter gene，NRT1.1B(OsNPF6.5)，may contribute to this divergence in nitrate use.Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica.NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes.The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication.Notably，field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency(NUE)compared to the variety without that allele.Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

科学家发明单碱基分辨率测序技术:CeU-Seq

近日，北京大学生命科学学院伊成器研究组报道了一种通过化学标记和富集手段实现全转录组水平上假尿嘧啶RNA修饰的单碱基分辨率测序技术CeU-Seq，并绘制了人和小鼠细胞转录组中假尿嘧啶RNA修饰的谱图。该研究进一步确定了多个可以作用于mRNA上的假尿嘧啶合成酶(其中PUS1、DKC1两种酶之前被发现与线粒体肌病、先天性角化不良等人类疾病相关)，并且发现转录组中假尿嘧啶的含量与分布均会受到各种环境刺激的调控，呈现出“刺激条件特异性”的诱导修饰。该研究为假尿嘧啶转录后修饰参与基因表达调控的研究提供了重要工具，为近年来兴起的“RNA表观遗传学”领域提供了崭新的研究方向。

论文链接:Li X，et al..Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome.

Nature Chemical Biology，2015，doi:10.1038/nchembio.1836.Published online:15 June，2015.

Abstract:Pseudouridine(Ψ)is the most abundant post-transcriptional RNA modification，yet little is known about its prevalence，mechanism and function in mRNA.Here，we performed quantitative MS analysis and show that Ψ is much more prevalent(Ψ/U ratio～0.2～0.6%)in mammalian mRNA than previously believed.We developed N3-CMC-enriched pseudouridine sequencing(CeU-Seq)，a selective chemical labeling and pulldown method，to identify 2 084 Ψ sites within 1 929 human transcripts，of which four(in ribosomal RNA and EEF1A1 mRNA)are biochemically verified.We show that hPUS1，a known Ψ synthase，acts on human mRNA;under stress，CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation.Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation.Collectively，our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of Ψ-mediated epigenetic regulation.

Crispr-Cas9技术获重大新成果

来自麻省总医院的一个研究小组找到了一种新方法来扩大强大基因编辑工具——Crispr-Cas9 RNA引导核酸酶的使用及提高其精确性。相比于迄今为止使用的自然形式的Cas9，演化版本的Cas9能够识别前者无法靶向的不同范围的核酸序列。采用该小组设计改进的新的Cas9变体，可以靶向过去用野生型Cas9无法改造的人类和斑马鱼基因。这将使得研究人员能够靶向各种基因组中更大范围内的一些位点，这个新方法可以用于需要高度精确靶向DNA序列的研究。该研究第一次证实了可以通过定向的蛋白质演化来改变SpCas9的活性，通过相似的方法还可以改变Cas9酶其他的有用特性，使得定制化一些重要特性成为可能。

论文链接:Kleinstiver B P，et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Nature，2015，doi:10.1038/nature14592.Published online:22 June，2015.

Abstract:Although CRISPR-Cas9 nucleases are widely used for genome editing，the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif(PAM).As a result，it can often be difficult to target double-stranded breaks(DSBs)with the precision that is necessary for various genome-editing applications.The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation.Here we show that the commonly used Streptococcus pyogenes Cas9(SpCas9)can be modified to recognize alternative PAM sequences using structural information，bacterial selection-based directed evolution，and combinatorial design.These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9，and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis.In addition，we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells，possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers.We also find that two smaller-size Cas9 orthologues，Streptococcus thermophilus Cas9(St1Cas9)and Staphylococcus aureus Cas9(SaCas9)，function efficiently in the bacterial selection systems and in human cells，suggesting that our engineering strategies could be extended to Cas9s from other species.Our findings provide broadly useful SpCas9 variants and，more importantly，establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

科学家开发出简单便宜的组织蛋白分析新技术

近日，来自瑞典乌普萨拉大学的研究人员开发了一种蛋白质分析技术，利用这种技术不需要高级设备、专门的实验室以及昂贵的试剂就可对组织蛋白进行分析。该技术以两个抗体对同一蛋白两个不同位点或两个定位很近的不同蛋白的结合为基础，将两个抗体分别与一段含发卡结构的DNA链连接，当两个抗体靠得非常近的时候，其带有的DNA链会结合形成一个起始序列。当这种情况发生时，通过杂交链式反应(hybridization chain reaction)将带有荧光标记并具有发卡结构的寡聚核苷酸连接到起始序列上，这样扩增出的每一条DNA链上都连接有荧光底物，实现了信号扩增，当使用特定波长的光进行激发，DNA链上连接的荧光底物会发射出荧光。当链式反应进行到一定程度，便可以在荧光显微镜下观察到明亮的点状荧光，点状荧光越多，蛋白就越多。除此之外，这种链式反应不需要任何酶的催化，在室温情况下就可进行反应。这项技术将给基础研究和医学诊断带来极大便利。

论文链接:Koos B，et al.Proximity-dependent initiation of hybridization chain reaction.

Nature Communications，2015，6:7294.doi:10.1038/ncomms8294.

Abstract:Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes.A method for this，which could be used in point-of-care devices and high-throughput screening，should be reliable，cost effective and robust.To achieve this，here we design a method(proxHCR)that combines the need for proximal binding with hybridization chain reaction(HCR)for signal amplification.When two oligonucleotide hairpins conjugated to antibodies bind in close proximity，they can be activated to reveal an initiator sequence.This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins，generating a fluorescent product.In conclusion，we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry.As no enzymes are needed，proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

研究发现检测血液中miRNA的简便技术

最近，密歇根大学的研究人员开发出一种有效的方法，能在血液中检测到癌变肿瘤脱落的microRNAs。这种方法可通过一种廉价的血液测试，同时对多种类型的癌症进行筛选——最终可能超过100种不同的类型。在实验中，研究人员用称为“捕获探针”的分子包覆一个载玻片，这些探针分子可紧紧抓住在其附近的microRNAs。这种方法的独特之处在于，DNA和RNA结合太弱，因此它们不能停留。虽然之前有研究人员已经在血清中检测到microRNA，但是本研究中的这种方法更为直接，并且几乎没有假阳性。

论文链接:Johnson-Buck A，et al.Kinetic fingerprinting to identify and count single nucleic acids.

Nature Biotechnology，2015，33(7):730－732.doi:10.1038/nbt.3246.Published online:22 June，2015.

Abstract:MicroRNAs(miRNAs)have emerged as promising diagnostic biomarkers.We introduce a kinetic fingerprinting approach calledsingle-molecule recognition through equilibrium Poisson sampling(SiMREPS)for the amplification-free counting of singleunlabeled miRNA molecules，which circumvents thermodynamic limits of specificity and virtually eliminates false positives.We demonstrate highconfidence，single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biofluids，as well as ＞500-fold discrimination between single nucleotide polymorphisms.