酿酒葡萄赤霞珠果实苹果酸生物合成关键基因的功能鉴定

针对近些年来葡萄果实有机酸含量受全球气候变暖影响而严重不足,酿造的葡萄酒风味和口感欠佳等问题,以宁夏地区栽培面积最多的赤霞珠(Cabernet Sauvignon)酿酒葡萄品种为材料,从果实形成至成熟4个生长时期(每个时期2次采样)的果实中提取RNA进行转录组测序,同时对同一时期的果实中与苹果酸生物合成关系密切的8种有机酸进行测定,通过基因表达量与有机酸含量的关联分析,筛选出影响赤霞珠果实中苹果酸生物合成途径的8种关键基因,并对其进行分离克隆、原核细胞表达和体外酶活性检测,从而确定各个关键基因及其同源基因的序列变化和功能差异,为今后酿酒葡萄有机酸含量的遗传调控、优质酿酒葡萄的培育提供帮助。     

‘金冠’苹果及其优系(SGP-1)果实发育期间有机酸代谢特征比较北大核心CSCD

以‘金冠’苹果及其优系‘SGP-1’为试材,测定果实发育期间有机酸组分、含量和苹果酸代谢相关酶活性,分析它们的变化规律及相关关系,以探索苹果有机酸积累的关键时期和关键酶,揭示果实低酸成因。结果表明:(1)苹果果实发育期间,‘SGP-1’的有机酸含量显著低于‘金冠’,成熟时仅为‘金冠’的二分之一,且主要由苹果酸、奎宁酸、酒石酸和柠檬酸组成,幼果期以奎宁酸为主,成熟期以苹果酸为主。(2)‘SGP-1’的苹果酸含量显著低于‘金冠’,其在幼果期和膨大期变化规律与‘金冠’相反,且积累关键时期和快速下降期早于‘金冠’;‘SGP-1’果实其余酸组分含量变化趋势与‘金冠’基本一致,但在幼果期显著高于‘金冠’,在成熟期与‘金冠’差异不显著。(3)‘SGP-1’的苹果酸代谢相关酶活性在幼果期均显著高于‘金冠’,成熟期持平或显著低于‘金冠’;幼果期MDH活性和成熟期ME活性在两材料间变化规律相反。(4)‘SGP-1’的幼果期苹果酸积累与PEPC和VHA活性呈极显著正相关关系,而同期‘金冠’则与MDH、PEPC和VHA活性呈极显著负相关关系;‘SGP-1’膨大期苹果酸积累与MDH、PEPC活性呈极显著负相关关系,与PEPCK和VHA活性呈显著正相关关系,而同期‘金冠’则与PEPC、ME和VHP活性呈极显著或显著负相关关系;二者成熟期苹果酸积累均与MDH、PEPC、ME和VHP活性呈极显著或显著负相关关系。研究发现,‘SGP-1’是以苹果酸为主的低酸型‘金冠’苹果变异优系,对苹果酸积累起主要调控作用的酶种类和活性与‘金冠’不同,导致了‘SGP-1’的低酸品质,该研究结果为深入探索果实低酸形成机理和培育高糖低酸新品种奠定了基础。     

‘京红’桃芽变缝合线局部早熟分子机制的探究北大核心CSCD

为了探究桃缝合线局部早熟的分子机制,该研究以‘京红’桃芽变(JHM)及其野生型(JHW)的果实为试材,测定分析了缝合线和果面部位的硬度、花青素含量以及差异基因的表达特征。结果显示:(1)‘京红’桃芽变比其野生型果实晚成熟约2周,芽变果实缝合线部位比果面部位局部早成熟,且提前2周时间转为红色。(2)随着果实的成熟,‘京红’桃野生型及其芽变的果实硬度逐渐降低,花青素含量逐渐升高,并均在花后66 d发生明显变化,芽变缝合线部位硬度比果面部位更低,花青素含量比果面更高。(3)在花后66 d,芽变果实的缝合线与果面部位差异表达基因数高达1889个,显著富集在代谢途径、次生代谢产物的生物合成、植物激素信号转导、苯丙素的生物合成等代谢途径;从中筛选到24个缝合线早熟相关基因,包含5个细胞壁降解相关基因,9个色素合成、调控相关基因,5个乙烯合成与转导相关基因,3个生长素应答基因和2个NAC转录因子。(4)对24个早熟相关基因中的12个差异表达基因进行荧光定量验证结果表明,基因表达趋势与转录组测序结果相一致。研究发现,桃芽变果实种仁产生的乙烯通过缝合线向周围扩散,促进缝合线部位ACS1和ACO1等基因的转录,并合成了较多乙烯,乙烯又进一步调控该部位PG、XTH33、CHS、DFR等细胞壁降解与色素合成相关基因的表达,导致该部位的果肉提前成熟。     

基于转录组测序的宁夏枸杞不同品种果实活性成分合成差异表达基因分析

为探究不同品种宁夏枸杞果实活性成分生物合成相关基因的表达水平,筛选关键差异表达基因(differentially expressed genes,DEGs),揭示宁夏枸杞品种间活性成分含量差异的分子机制,本研究采用Illumina NovaSeq 6000高通量测序技术,对宁夏枸杞‘宁杞1号’和‘宁杞7号’青果期、转色期及成熟期果实进行转录组测序,比较2个品种果实不同发育期相关基因表达谱的变化。结果显示:转录组测序共获得811818178条clean reads,有121.76 Gb有效数据。‘宁杞1号’和‘宁杞7号’在青果期、转色期和成熟期差异表达基因分别有2827、2552和2311个;分别有2153、2050和1825个差异基因在基因本体论(gene ontology,GO)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析和同源蛋白簇(clusters of orthologous groups of proteins,KOG)分析等6个数据库中被成功注释。青果期、转色期和成熟期果实的差异表达基因,在GO数据库分别有1307、865和624个被富集到生物学过程、细胞组分及分子功能3个部分中;KEGG通路富集结果均集中在代谢途径、次生代谢物生物合成和植物-病原互作过程;在KOG数据库,3个发育期分别注释了1775、1751和1541个差异表达基因。对注释的基因进行PubMed数据库检索,在青果期、转色期和成熟期分别筛选到与枸杞活性成分合成相关的差异表达基因18、26和24个,这些基因主要参与类胡萝卜素、类黄酮、萜类、生物碱和维生素等代谢途径。选取7个差异表达基因进行RT-qPCR验证,结果与转录组测序数据表达趋势一致。本研究从转录水平为不同品种宁夏枸杞活性成分含量差异提供了初步证据,为进一步挖掘枸杞活性成分生物合成的关键基因及解析其表达调控机制提供了研究基础。     

不同鲜食品质橄榄果实转录组测序及酚类代谢途径相关调控基因挖掘

【目的】果实酚类物质类别、含量是与橄榄营养及风味密切相关的重要品质性状,探究橄榄酚类物质生物合成的分子调控机制。【方法】以总酚含量差异显著的橄榄（低酚/高酚）为试材,分别取花后80-160 d的果实进行转录组分析,对莽草酸-水解单宁/苯丙烷-类黄酮生物合成通路差异基因进行表征,并对差异表达基因进行WGCNA分析,从中挖掘与酚类代谢途径相关的转录因子。【结果】转录组测序共获得296 314条Unigene,其中73%的Unigene被注释到数据库;4个品种（系）橄榄成熟果共鉴定到1 628个差异表达基因（DEGs）,KEGG分析显示,DEGs在酚类代谢途径的“类黄酮生物合成”通路上显著富集;进一步对果实成熟过程莽草酸-水解单宁/苯丙烷-类黄酮生物合成途径的DEGs进行表征,结合WGCNA分析中每个模块与性状的R2和P值,筛选到4个关键模块,根据模块内基因的调控关系利用MCC拓扑分析法以度值≥1挖掘模块内关键转录因子,挖掘到137个转录因子Unigene与30个酚类合成结构基因Unigene共表达,转录因子Unigene功能注释来自35个基因家族,最多的为锌指蛋白（C2C2、C3H、C2H...     

酿酒葡萄‘赤霞珠’VvbHLH 93基因的克隆及分析北大核心CSCD

bHLH93转录因子参与调节植物的生长发育、应对各种胁迫等多种生理过程,该研究以酿酒葡萄‘赤霞珠’为试验材料,采用RT-PCR方法克隆葡萄VvbHLH 93基因全长,并进行生物信息学分析;用qRT-PCR法分析bHLH93在不同组织和果实不同发育时期的表达量,为进一步探索VvbHLH 93基因的功能及其机制奠定基础。结果表明:(1)成功克隆获得葡萄VvbHLH 93,该基因cDNA全长为1319 bp,开放阅读框长度为939 bp,编码312个氨基酸,相对分子量为35.18 kD,理论等电点为4.68,无信号肽和跨膜域,属于bHLH转录因子家族。(2)葡萄bHLH93蛋白与荷花的亲缘关系较近;VvbHLH93蛋白的C端为酸性结构区,富含丝氨酸、苏氨酸磷酸化位点;VvbHLH 93基因的启动子含有光响应元件和低温、干旱、赤霉素等应答元件。(3)qRT-PCR分析表明,VvbHLH 93在‘赤霞珠’中的表达具有组织特异性,在叶片中基本不表达,在茎中的表达量最高;随着赤霞珠葡萄果实的发育成熟,VvbHLH93相对表达量不断降低,到幼果期(直径>2 mm)后第5周开始相对表达量基本为0,总体呈现降低的变化趋势;与对照相比,在低温胁迫、盐胁迫、高温胁迫及充分灌溉胁迫下VvbHLH 93表达量显著降低。研究推测,VvbHLH 93基因可能是葡萄抗逆胁迫的负转录因子。     

FaMYB73调控草莓果实苹果酸合成的研究

苹果酸是草莓果实主要的有机酸之一,其含量的高低显著影响草莓果实的风味。该研究以‘红颜’草莓为试材,采用反转录(RT PCR)的方法克隆得到一个新的MYB转录因子——FaMYB73。该基因包含有756 bp的开放阅读框,编码252个氨基酸,预测其蛋白质分子量为27.6 kD,等电点为6.8。功能域分析表明,FaMYB73蛋白含有保守的R2R3结构域;亚细胞定位于细胞核中。实时荧光定量(qRT PCR)分析表明,FaMYB73基因在草莓各个组织均有表达,且在花和叶中的表达相对较高;在果实的白果期FaMYB73基因的表达量最高,且随着果实成熟,FaMYB73基因的表达下调,说明该基因的表达具有组织和时期特异性。高效液相色谱法(HPLC)检测结果发现,苹果酸含量在草莓果实发育期呈先上升后下降的趋势,且与FaMYB73基因的表达量呈正相关。草莓瞬时注射结果显示,FaMYB73过表达显著促进了苹果酸的积累,沉默则显著降低了苹果酸的含量。同时,获得了2个转FaMYB73基因烟草稳定表达株系,FaMYB73过量表达显著提高了烟草叶片中苹果酸的含量。研究表明,FaMYB73参与调控草莓果实中苹果酸的合成,为进一步研究其调控机制提供了理论依据。     

河西走廊不同产地‘赤霞珠’酿酒葡萄果实品质评价

为了确定河西走廊地区‘赤霞珠’酿酒葡萄核心品质指标,建立‘赤霞珠’酿酒葡萄品质综合评价模型。从张掖、武威、嘉峪关3个酿酒葡萄主产市的代表性果园采集6份‘赤霞珠’葡萄样品进行品质测定,通过主成分分析和聚类分析法确定‘赤霞珠’葡萄核心品质指标,运用层次分析法确定指标权重并建立‘赤霞珠’葡萄品质综合评价模型。结果表明:(1)不同产地‘赤霞珠’酿酒葡萄品质指标存在明显差异性,张掖市国风葡萄酒庄园的‘赤霞珠’葡萄果糖、蔗糖、草酸、柠檬酸含量均高于其他地区,且可溶性固形物、可溶性糖、总酚、苹果酸含量在各产地中也均保持在最高水平。(2)相关性分析发现,葡萄果实葡萄糖含量与可溶性固形物含量、果糖与可溶性糖含量之间呈极显著正相关,固酸比和糖酸比均与可滴定酸含量呈极显著负相关关系。(3)综合主成分分析、聚类分析和相关性分析结果,确定维生素C(Vc)、单宁、果糖和固酸比是‘赤霞珠’葡萄核心品质指标,应用层次分析法建立了‘赤霞珠’葡萄品质综合评价模型为Y=0.0960×Vc含量+0.1611×单宁含量+0.2771×果糖含量+0.4568×固酸比(各指标含量均经过标准化处理)。研究发现,河西走廊地区‘赤霞珠’葡萄果实品质最佳产地是张掖市,果实Vc、单宁、果糖和固酸比是‘赤霞珠’葡萄的核心品质指标,以其建立的评价模型可用于‘赤霞珠’葡萄品质的综合评价。     

SlCBL1基因在番茄抗灰霉病中的作用

以番茄(Solanum lycopersicum L.)品种‘Micro Tom’为试材,分析番茄叶片和果实的灰霉病发病规律,及番茄类钙调磷酸酶B基因(tomato calcineurin B-like gene,SlCBL1)在叶片和果实中的表达变化;比较转SlCBL1基因番茄与对照的叶片和果实的灰霉病发病过程,分析转基因番茄抗病相关转录因子表达变化。结果表明:(1)非转基因番茄中,不同叶龄的叶片均在接种灰霉病4d开始发病;不同发育阶段的果实接种灰霉病后发病时间也不同,其中绿果(花后16~18d)接种5d还未发病,白果(花后34~36d)接种11d开始发病,红果(花后40~42d)接种5d开始发病;SlCBL1基因表达量在番茄叶片中较低,在绿果期和白果期的果实中表达量最高,红果期果实中表达量最低。(2)转SlCBL1基因后,SlCBL1基因的过量表达能够抑制番茄叶片和果实灰霉病发生;同时番茄叶片和果实中几乎所有的抗病转录因子的表达量都上调,其中WRKY转录因子家族基因SlWRKY33和SlWRKY70受到强烈调控。研究说明,SlCBL1基因过量表达能够提高番茄的抗灰霉能力,其主要机理是通过影响抗病相关转录因子进而调控番茄抗灰霉病的能力。     

ABA信号通路对葡萄果皮花青苷生物合成的调控机制研究

以‘红巴拉多’葡萄为试验材料,在转色前期(约花后6周)用300 mg/L的ABA对果穗进行处理,以清水处理为对照;测定不同发育时期葡萄果实的单果重、可滴定酸、可溶性固性物等生理指标,同时测定果皮中总花青苷及ABA含量;检测不同发育时期果皮中ABA信号通路和花青苷生物合成相关基因表达量,克隆6个与花青苷生物合成相关基因的启动子,并预测启动子中的顺式作用元件,在转录调控水平上探讨ABA信号通路对葡萄果皮花青苷生物合成的调控作用。结果表明:(1)ABA处理的葡萄果实可溶性固形物含量明显提高、可滴定酸含量下降。(2)ABA处理显著提高了‘红巴拉多’葡萄果皮的着色水平以及总花青苷和ABA含量。(3)ABA处理后,9个ABA信号通路基因以及6个花青苷生物合成相关基因表达水平明显提高。(4)6个花青苷生物合成相关基因的启动子序列中均含有多个与ABA响应相关的ABRE作用元件。研究发现,9个ABA信号通路基因可能在葡萄果皮着色中发挥着重要作用,其中2个VvABFs转录因子可能直接作用于含有ABRE元件的花青苷生物合成相关基因的启动子序列,推测可通过调控这些基因的转录水平来调控葡萄果皮花青苷的积累。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.