南方红豆杉毛状根诱导体系的建立及毛状根中 紫杉醇的分离纯化研究

目的:研究发根农杆菌对南方红豆杉转化的转化体系和毛状根中紫杉醇的提取纯化工艺;方法:采用发根农杆菌对南方红豆杉不同外植体进行诱导,并对影响毛状根转化的因素进行分析。采用超临界CO2流体萃取法、硅胶柱色谱法以及高效液相法对毛状根中的紫杉醇进行分离提纯;结果:成功构建了南方红豆杉毛状根诱导体系。其中农杆菌的种类、外植体类型、预培养和共培养时间、培养基中激素浓度均影响毛状根的转化率。毛状根经高压纸电泳检测显示表达冠瘿碱。毛状根中紫杉醇经超临界CO2法提取及硅胶柱色谱法分离提纯,采用高效液相法分析显示其纯度可达到98%。结论:发根农杆菌诱导南方红豆杉产生的毛状根中可产生紫杉醇,可作为紫杉醇的主要来源。     

宁夏枸杞发根农杆菌转化系的建立及影响转化因素的研究

以发根农杆菌Ａ４菌株介导,对宁夏枸杞叶片和茎切段的遗传转化进行了初步研究,建立了发状根体系,并优化了转化条件。乙酰丁香酮的添加、农杆菌液的浓度、共培养时间、外植体取材部位及时间,均可以影响发状根的诱导频率。采用添加１００μｍｏｌ／Ｌ的 乙酰丁香酮、振荡培养２４ｈ的Ａ４菌液感染３周龄的叶片切段,并共培养３ｄ,可以得到最佳的转化效果,发状根的诱导率为４８．９％。在同样的转化条件下,只有１３．６％的茎切     

新疆杨高效遗传转化系统的建立

选择新疆杨(Populus alba L.var．pyramidalis Bge．)为遗传转化受体材料,为建立根癌农杆菌介导新疆杨高效遗传转化系统,从预培养时间、侵染时间、共培养时间、添加乙酰丁香酮(AS)的时机、共培养培养基中添加乙酰丁香酮浓度、侵染菌液的制备方法、外植体继代方式等7个方面优化筛选。结果显示较合适的转化系统为：预培养8h,农杆菌菌液(OD600=0．4)侵染15min,共培养5d,侵染菌液的最优制备方法是液体培养活化农杆菌2次加离心收集菌体重悬,共培养培养基中添加乙酰丁香酮80μmol／L。新疆杨叶盘转化频率可达38．10％。     

外植体龄和蔗糖浓度对黄瓜子叶产生毛状根的影响

研究了外植体龄和蔗糖浓度对发根农杆菌 R160 1介导黄瓜子叶产生毛状根的影响。结果表明 :以 10 d龄子叶外植体产生毛状根的能力最强 ,外植体的毛状根诱导率为 88.89% ;2 0 d龄子叶外植体的毛状根诱导率比 10 d龄子叶外植体降低 52 .86% ;30 d龄子叶外植体感染发根农杆菌R160 1后不产生毛状根。感染发根农杆菌 R160 1的黄瓜子叶外植体在不加或加 1%蔗糖的 MS培养基上的毛状根诱导率极低 ,子叶外植体逐渐变黄 ,腐烂 ;而培养基中添加 2 % ,3%或 4 %的蔗糖可显著提高子叶外植体的毛状根诱导率。黄瓜毛状根能在无外源植物激素的 MS液体培养基中自主生长。冠瘿碱的高压纸电泳检测表明毛状根已被 Ri T- DNA转化     

发根农杆菌诱导桑树毛状根体系的建立

应用发根农杆菌ACCC10060,以直接接种和共培养2种方法侵染桑树10 d龄子叶,并将2种处理的外植体分别接种于MS+AS(乙酰丁香酮,100 μmol/L)的平板,暗培养2 d后转接至MS+CS(头孢霉素,200 mg/L)平板培养3周,每3 d转接1次以除去其中所含的发根农杆菌菌体,结果2种侵染方法均成功诱导桑树产生毛状根,诱导效率分别为14%和17%.在无激素MS培养基上离体培养除菌后的毛状根,呈现旺盛的生长态势和典型的发状根结构特点.CTAB法提取毛状根基因组并进行PCR检测,结果扩增出了423 bp的rolB基因片段,表明Ri质粒的T-DNA已经成功整合到桑树的基因组中.     

木豆毛状根的诱导及其培养条件的研究

利用发根农杆菌LBA9402对木豆叶片直接进行诱导产生毛状根。本实验研究出诱导木豆毛状根的最佳条件是,以木豆叶片为外植体,于1/2MS固体培养基上预培养2~4 d,菌液浓度OD₆₀₀=0.6~0.8,浸染20 min,共培养3 d,诱导率为60.00%。在分子水平用PCR检测表明,发根农杆菌9402Ri质粒上的T-DNA成功整合进木豆毛状根的基因组中。     

极性和NAA浓度对发根农杆菌遗传转化黄瓜子叶的影响

本文研究了极性和NAA浓度对发根农杆菌（Agrobacteriumrhizolgenes）R1000和R1601诱导黄瓜子叶产生毛状根的影响。结果表明,感染发根农杆菌R1000和R1601的黄瓜子叶都仅在其下端产生毛状根。培养基中加人0．1mgL-1和5．0mgL-1NAA预培养和共培养都可提高发根农杆菌R1000和R1601对黄瓜子叶外植体下端的生根能力。但仅用5．0mgL-1NAA预培养和共培养的感染发根农杆菌R1000和R1601的子叶外植体上端可不同程度地生根,生根率分别为32．5％和6．48％。经检测,毛状根均含有农杆碱和甘露碱。     

4种发根农杆菌对朱砂根组培无菌叶片毛状根诱导的影响

以朱砂根(Ardisia crenata Sims)组培无菌叶片为材料,用4种发根农杆菌菌株(A4、ATCC15834、LBA9402和R1601)分别侵染进行毛状根诱导,比较朱砂根叶片毛状根诱导的最适培养基种类、预培养时间、侵染方式、共培养时间以及不同发根农杆菌的致根能力。研究表明:(1)朱砂根无菌叶片毛状根诱导最适培养基为1/2MS培养基,预培养2d、共培养2d,毛状根诱导率最高(31.87%)。(2)最佳侵染方式以剪好的幼叶和活化好的菌液(100mg/L AS)一起在28℃、180r/min黑暗条件下共振荡8~15min。(3)4种发根农杆菌均能诱导朱砂根叶片毛状根产生,但A4、ATCC15834效果最好,其致根能力大小顺序依次为ATCC15834A4LBA9402R1601。(4)PCR分子鉴定表明,发根农杆菌Ri质粒T-DNA已成功整合到宿主细胞核基因组中。     

滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。     

发根农杆菌介导的长春花高效转基因体系的建立

以长春花幼叶为外植体建立了发根农杆菌介导的长春花高效遗传转化体系,主要技术环节为：用携带有基因表达载体的发根农杆菌R1000侵染幼嫩叶片,侵染的叶片外植体与发根农杆菌共培养2d,外植体移至除菌培养基除菌培养2～3周,切取外植体上诱导长出的毛状根置于筛选培养基上培养1-2周,最后对筛选出的阳性毛状根无性系进行扩繁。筛选出的阳性毛状根经GUS染色和PCR分子鉴定表明,该方法的发根诱导率和阳性转化率分别为82％±2．49％和100％。该转化方法所获得的毛状根系数量大、质量高、遗传稳定且所需时间短,明显优于现有的长春花遗传转化技术,是长春花遗传转化的高效便捷体系。     

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.