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[学术文献] Crystal structures of rice hexokinase 6 with a series of substrates shed light on its enzymatic mechanism 进入全文

Biochemical and Biophysical Research Communications

Hexokinases (HXKs) have determined to be multifaceted proteins, and they are the only ones able to phosphorylate glucose in plants. However, the binding mode for ATP to plant HXK5 remains unclear. Here, we report the crystal structures of rice hexokinase 6 (OsHXK6) in four different forms: (i) apo-form, (ii) binary complex with D-Glc, (iii) quaternary complex with ADP, PO4 and Mg2+, and (iv) pentanary complex with D-Glc, ADP, PO4, and Mg2+. The apo form is in the open state conformation, and the three others are in the closed state, indicating that glucose and ADP-PO4 binding induces a large conformational change by domain rearrangement. The quaternary complex is a novel intermediate during the catalytic reaction we trapped for the first time, which provides a new evidence for the enzymatic mechanism of HXKs. In addition, the latter two complexes reveal the binding mode for ADP-PO4 to plant HXK5, which provide the structural explanation for the dual-function of OsHXK6. In addition, we identified that residues G1y112, Thr261, G1y262, and G1y450 are essential to the binding between ADP-PO4 and OsHXK6 by a series of single mutations and enzymatic assays. Our study provide structural basis for the other functional studies of OsHXK6 in rice. (C) 2019 Published by Elsevier Inc.

[前沿资讯] 武汉大学红莲型水稻品鉴会推荐最佳品种在鄂推广种植 进入全文

中国产经新闻网

近日,武汉大学杂交水稻国家重点实验室与湖北省粮食行业协会联合举办红莲型水稻新品种食味品鉴会,来自湖北省粮协、国宝桥米、什湖知音、湖北洪森等11位业内专家对10个红莲型水稻新品种进行打分品评,推荐最佳品种在湖北省推广种植。红莲型杂交水稻由武汉大学朱英国院士团队积40年之力培育,是国际公认的杂交水稻三大细胞质类型之一,是我国杂交水稻重要的遗传资源。它既可在我国长江流域用作中稻,华南稻作区用作早、中、晚稻种植,也适合在南亚、东南亚和非洲国家推广种植。  

[前沿资讯] 作物基因组编辑育种技术方法研究中取得进展 进入全文

中国科学院

遗传与变异是物种进化的基础。通过物理、化学方法(如辐射诱变、EMS诱变)产生全基因组的随机突变已经成为农作物育种的常规手段,但其中具有新型农艺性状突变体的筛选较为费时、费力。定向进化(Directed Evolution)则通过创制目标基因的突变文库,在施加一定选择压力下能够快速获得目的突变体。目前,植物基因的定向进化通常先通过易错PCR、DNA合成或DNA重组等方法在体外产生目标基因的突变文库,再转化到大肠杆菌或酵母中进行功能筛选。然而,由于离开原始的基因组和细胞环境,筛选出来的基因突变可能并不能完全反映出它在植物中的真实功能。更重要的是,大多数重要农艺性状无法在大肠杆菌或酵母中进行筛选。因此,建立一种在植物原位进行基因饱和突变和功能筛选的定向进化新方法将有助于加快植物育种及重要功能基因研究的进程。近日,中国科学院遗传与发育生物学研究所高彩霞研究组和李家洋研究组合作,构建了新型的饱和靶向内源基因突变碱基编辑器STEME,并在植物中实现了基因的定向进化和功能筛选。研究者将胞嘧啶脱氨酶APOBEC3A和腺嘌呤脱氨酶ecTadA-ecTadA7.10同时融合在nCas9 (D10A)的N端,并将抑制体内尿嘧啶糖基化酶UDG的活性的UGI以融合或自由表达的形式置于nCas9 (D10A)的C端,共构建了4种形式的双碱基编辑器STEME(STEME-1至STEME-4)。STEME双碱基编辑器均可以只在一个sgRNA引导下就可以诱导靶位点C>T和A>G的同时突变,显著增加了靶基因碱基突变的饱和度及产生突变类型的多样性。STEME-1在水稻原生质体中C>T诱导效率高达61.61%,C>T和A>G同时突变的效率也高达15.50%。为了提高靶基因的碱基覆盖度,研究者进一步利用能够识别NG PAM的变体Cas9-NG构建了第5个双碱基编辑器STEME-NG,发现只需要20个sgRNA就可以对OsACC上编码56个氨基酸的序列实现近饱和的突变。为了展示STEME在植物中的定向进化能力,研究者设计了靶向OsACC羧基转移酶结构域上400个氨基酸编码序列的200个独立的sgRNA,分别构建到STEME-1或STEME-NG双元载体上。将构建好的双元载体分为27个转化组,每个组内混合了等分子量的4至11个sgRNA载体,覆盖80-142 bp的靶DNA区域,便于提高转化效率和突变的高通量测序。经农杆菌介导法转化水稻愈伤,共获得约6000株水稻再生苗。水培法炼苗10天后,对再生苗喷洒高效氟吡甲禾灵(Haloxyfop)进行筛选,共发现四个除草剂抗性突变位点:P1927F、W2125C、S1866F和A1884P。除W2125C以外,其余三个抗性位点未曾在植物中有过报道。其中,P1927F与W2125C突变一样表现出强除草剂抗性,具有较高的生产应用潜能。基于同源蛋白结构模型分析发现,这些氨基酸突变直接或间接地影响了除草剂结合口袋的构象,从而降低了其对除草剂分子的结合能力而获得除草剂抗性。STEME技术体系的建立,对于原位(In situ)定向进化植物的内源功能基因提供了新型技术支撑,对农作物分子设计育种具有重要意义。此外,STEME系统还有望应用于不同细胞系、酵母或动物中的非编码区的顺式作用元件的调控、动物致病SNV的修正和抗药位点的筛选等。该研究成果于2020年1月13日在线发表于《自然-生物技术》(Nature Biotechnology)杂志(DOI:10.1038/s41587-019-0393-7)。高彩霞研究组博士生李超、助理研究员张瑞以及李家洋研究组副研究员孟祥兵为本文共同第一作者,研究员高彩霞和李家洋为共同通讯作者。遗传发育所陈宇航研究组和中科院微生物研究所邱金龙研究组参与了部分研究工作。该研究得到中科院战略性先导专项、国家自然基金委等的经费资助。

[前沿资讯] 我国科学家创出水稻基因组定点编辑新办法 进入全文

中国科学院

据中国农科院最新消息,该院植物保护研究所作物有害生物功能基因组研究创新团队,首次将ScCas9蛋白应用于水稻细胞基因位点识别和定点编辑,证实在水稻中有较高的编辑效率,可识别PAM序列NNG基因位点,扩宽了植物基因编辑应用范围。相关研究成果在线发表于《植物生物(Plant Biotechnology Journal)》。中国农科院植保所研究员周焕斌介绍,CRISPR/Cas9系统已成为基因组精准修饰的有效工具,促进了植物功能基因组学研究和作物分子育种进程。但是Cas蛋白仅识别特定的PAM序列,极大的局限了基因编辑,尤其是碱基编辑靶点的选择性。利用Cas蛋白突变体和不同物种Cas蛋白等在一定程度上可扩宽CRISPR工具的打靶范围。扩展Cas9的识别区域是当前对CRISPR/Cas9系统优化改良的重要方向。该研究选用来源于狗链球菌的ScCas9蛋白对水稻基因组编辑技术进行升级和扩宽编辑范围。试验证实,ScCas9蛋白在水稻中可通过识别NNG位点完成基因编辑,且对NAG位点表现最好,编辑效率优于与之相似(相似度89.2%)的传统SpCas9蛋白,但对NGG、NCG和NTG的编辑效率则具有一定的位点依赖性。此外,ScCas9蛋白还可同时高效地完成多基因编辑和双靶碱基定点替换,编辑效率高达36.96%和47.5%。ScCas9在水稻基因组定点编辑上的应用,扩宽了基因组编辑的范围,为后续基因组编辑衍生技术提供了更多的可选择工具,也为植物基因功能研究和作物分子育种与遗传改良提供了有力的技术支撑。

[学术文献] Synergistic Effects of Nanocomposite Films Containing Essential Oil Nanoemulsions in Combination with Ionizing Radiation for Control of Rice 进入全文

Journal of Food Science

The fumigant toxicity of eight individual essential oils (EOs; basil, cinnamon, eucalyptus, mandarin, oregano, peppermint, tea tree, and thyme) and one binary combination (thyme and oregano) for control of the rice weevil, Sitophilus oryzae, were investigated. In bioassays, all individual and combined EOs were toxic to the rice weevil. Eucalyptus EO exhibited the highest toxicity among the individual EO treatments, causing 100% mortality at a minimum concentration of 0.8 μL/mL after 24 hr of exposure. The combination treatment of oregano and thyme EO displayed higher fumigant activity than the individual oregano or thyme treatments. A stable oil-in-water nanoemulsion was evaluated using highpressure homogenization (microfluidization [MF]) and varying the pressure and number of cycles. The droplet size of the emulsions was found to decrease from 217 to 71 nm and encapsulation efficiency increased from 37% to 84% with increasing MF pressure and number of cycles. The optimum conditions for preparing the mixture of oregano and thyme EO nanoemulsions were evaluated to be homogenization pressure of 103 MPa and three cycles. Incorporating an oregano:thyme nanoemulsion (0.75%) into cellulose nanocrystal (CNC) containing chitosan (CH/CNC), methyl cellulose (MC/CNC), and polylactic acid (PLA/CNC) composite films resulted in extended diffusion matrices causing 32% to 51% rice weevil mortality after 14 days exposure. Irradiation at 200 Gray alone caused 79% mortality and increased to 100% when combined with the bioactive chitosan film containing the oregano:thyme nanoemulsion.

[学术文献] Genetic analysis of environment-sensitive genic male sterile rice under US environments 进入全文

Euphytica

Two line hybrid rice (Oryza sativa L.) breeding uses environment-sensitive genic male sterile (EGMS) lines to produce sterile or fertile pollen depending on daylength and/or temperature. There is limited information on the performance and genetic control of EGMS lines under U.S. environments. Therefore, genetic characterization of two F-2 and four BC1F2 populations derived from EGMS line 2009S was conducted under Louisiana field conditions. Chi squared analyses in the F-2 and BC1F2 populations indicated that pollen sterility under high temperature and long daylength field conditions was controlled by a single recessive gene. Sequence comparisons at locus LOC_Os02g12290 between 2009S, CL161 (USDA-AMS 2002) and published sequences of G63S and Nipponbare revealed a single nucleotide polymorphism (SNP) that has been detected previously in several EGMS lines. Due to high GC content, a CEL1 nuclease assay was used to detect SNPs associated with pollen sterility in 177 F-2 and 59 BC1F2 sampled individuals. A high percentage of lines (90-100%) across all segregating populations were identified correctly as pollen sterile using the CEL 1 assay. Results from this study suggest that single-gene control of pollen sterility in EGMS line 2009S will be compatible with a two-line system for U.S. hybrid rice development.

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