研究發(fā)現(xiàn)控制水稻粒形和稻米品質(zhì)的重要基因

近日，中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所傅向東研究員領(lǐng)導(dǎo)的團(tuán)隊從優(yōu)質(zhì)雜交水稻不育系泰豐A中成功分離并克隆了一個控制水稻粒形和提升稻米品質(zhì)的重要基因GW7。這一基因能通過改變細(xì)胞分裂模式，讓稻米變得更為細(xì)長，有效地減少堊白率和堊白面積，從而提高稻米在外觀、口感等方面的品質(zhì)。該研究還表明將GW7和GS3基因的優(yōu)異等位變異聚合并應(yīng)用到我國高產(chǎn)秈稻中，可明顯提高稻米品質(zhì)，同時還可提高產(chǎn)量。該項研究為水稻高產(chǎn)優(yōu)質(zhì)分子模塊設(shè)計育種提供了具有重要應(yīng)用價值的新基因，也為揭示水稻品質(zhì)和產(chǎn)量協(xié)同遺傳改良的分子奧秘提供了新線索。

論文鏈接: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

植物主要以銨態(tài)氮和硝態(tài)氮為主要氮源，中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所儲成才研究員領(lǐng)導(dǎo)的團(tuán)隊研究表明，秈稻品種利用硝酸鹽的能力顯著高于粳稻品種，該團(tuán)隊通過圖位克隆技術(shù)從秈稻中克隆出高氮利用效率基因NRT1.1B，NRT1.1B編碼一個硝酸鹽轉(zhuǎn)運蛋白，在秈、粳稻間只有一個氨基酸的差別，且秈稻與粳稻呈現(xiàn)出顯著的分化，各種證據(jù)表明，秈稻型具有更高的硝酸鹽吸收及轉(zhuǎn)運活性。尤為重要的是，秈稻中的硝酸鹽同化過程的關(guān)鍵基因也被顯著上調(diào)，這種結(jié)果導(dǎo)致秈稻具有更高的氮肥利用能力，說明NRT1.1B在粳稻氮肥利用效率改良上具有巨大應(yīng)用價值。

論文鏈接: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.

科學(xué)家發(fā)明單堿基分辨率測序技術(shù):CeU-Seq

近日，北京大學(xué)生命科學(xué)學(xué)院伊成器研究組報道了一種通過化學(xué)標(biāo)記和富集手段實現(xiàn)全轉(zhuǎn)錄組水平上假尿嘧啶RNA修飾的單堿基分辨率測序技術(shù)CeU-Seq，并繪制了人和小鼠細(xì)胞轉(zhuǎn)錄組中假尿嘧啶RNA修飾的譜圖。該研究進(jìn)一步確定了多個可以作用于mRNA上的假尿嘧啶合成酶(其中PUS1、DKC1兩種酶之前被發(fā)現(xiàn)與線粒體肌病、先天性角化不良等人類疾病相關(guān))，并且發(fā)現(xiàn)轉(zhuǎn)錄組中假尿嘧啶的含量與分布均會受到各種環(huán)境刺激的調(diào)控，呈現(xiàn)出“刺激條件特異性”的誘導(dǎo)修飾。該研究為假尿嘧啶轉(zhuǎn)錄后修飾參與基因表達(dá)調(diào)控的研究提供了重要工具，為近年來興起的“RNA表觀遺傳學(xué)”領(lǐng)域提供了嶄新的研究方向。

論文鏈接: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技術(shù)獲重大新成果

來自麻省總醫(yī)院的一個研究小組找到了一種新方法來擴(kuò)大強(qiáng)大基因編輯工具——Crispr-Cas9 RNA引導(dǎo)核酸酶的使用及提高其精確性。相比于迄今為止使用的自然形式的Cas9，演化版本的Cas9能夠識別前者無法靶向的不同范圍的核酸序列。采用該小組設(shè)計改進(jìn)的新的Cas9變體，可以靶向過去用野生型Cas9無法改造的人類和斑馬魚基因。這將使得研究人員能夠靶向各種基因組中更大范圍內(nèi)的一些位點，這個新方法可以用于需要高度精確靶向DNA序列的研究。該研究第一次證實了可以通過定向的蛋白質(zhì)演化來改變SpCas9的活性，通過相似的方法還可以改變Cas9酶其他的有用特性，使得定制化一些重要特性成為可能。

論文鏈接: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.

科學(xué)家開發(fā)出簡單便宜的組織蛋白分析新技術(shù)

近日，來自瑞典烏普薩拉大學(xué)的研究人員開發(fā)了一種蛋白質(zhì)分析技術(shù)，利用這種技術(shù)不需要高級設(shè)備、專門的實驗室以及昂貴的試劑就可對組織蛋白進(jìn)行分析。該技術(shù)以兩個抗體對同一蛋白兩個不同位點或兩個定位很近的不同蛋白的結(jié)合為基礎(chǔ)，將兩個抗體分別與一段含發(fā)卡結(jié)構(gòu)的DNA鏈連接，當(dāng)兩個抗體靠得非常近的時候，其帶有的DNA鏈會結(jié)合形成一個起始序列。當(dāng)這種情況發(fā)生時，通過雜交鏈?zhǔn)椒磻?yīng)(hybridization chain reaction)將帶有熒光標(biāo)記并具有發(fā)卡結(jié)構(gòu)的寡聚核苷酸連接到起始序列上，這樣擴(kuò)增出的每一條DNA鏈上都連接有熒光底物，實現(xiàn)了信號擴(kuò)增，當(dāng)使用特定波長的光進(jìn)行激發(fā)，DNA鏈上連接的熒光底物會發(fā)射出熒光。當(dāng)鏈?zhǔn)椒磻?yīng)進(jìn)行到一定程度，便可以在熒光顯微鏡下觀察到明亮的點狀熒光，點狀熒光越多，蛋白就越多。除此之外，這種鏈?zhǔn)椒磻?yīng)不需要任何酶的催化，在室溫情況下就可進(jìn)行反應(yīng)。這項技術(shù)將給基礎(chǔ)研究和醫(yī)學(xué)診斷帶來極大便利。

論文鏈接: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.

研究發(fā)現(xiàn)檢測血液中miRNA的簡便技術(shù)

最近，密歇根大學(xué)的研究人員開發(fā)出一種有效的方法，能在血液中檢測到癌變腫瘤脫落的microRNAs。這種方法可通過一種廉價的血液測試，同時對多種類型的癌癥進(jìn)行篩選——最終可能超過100種不同的類型。在實驗中，研究人員用稱為“捕獲探針”的分子包覆一個載玻片，這些探針分子可緊緊抓住在其附近的microRNAs。這種方法的獨特之處在于，DNA和RNA結(jié)合太弱，因此它們不能停留。雖然之前有研究人員已經(jīng)在血清中檢測到microRNA，但是本研究中的這種方法更為直接，并且?guī)缀鯖]有假陽性。

論文鏈接: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.