新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

冬凌草毛状根的优化培养及其提取物的肿瘤细胞增殖抑制活性检测

探究并建立冬凌草毛状根的优化培养方案并检测其提取物的抗肿瘤活性。用发根农杆菌ATCC11325及ATCC15834诱导冬凌草外植体生成毛状根,并探究培养基种类、蔗糖浓度等条件对冬凌草毛状根生长的影响,测定其生长曲线;超声法制备毛状根粗提物,大孔吸附树脂进一步纯化粗提物获得提取物,用高效液相色谱法检测冬凌草甲素含量;提取物配制成不同浓度试液作用于癌细胞株BEL-7402、A-549、SGC-7901和HGC,用CCK-8法测定细胞存活率。仅ATCC15834可诱导冬凌草从叶柄和茎部切口处长出不定根,叶片外植体未见不定根萌发,不定根遗传鉴定符合毛状根特征。毛状根在含3%蔗糖B5培养基中增殖率最高,其生长曲线近似"S"型,25 d达到对数中期,70 d达到平台期,其冬凌草甲素含量分别为0.0171%、0.0022%。当毛状根提取物含量为1 mg/mL时,对四种癌细胞生长的抑制率均达95%以上。以ATCC15834侵染冬凌草叶柄和茎部外植体诱导出的冬凌草毛状根提取物在含3%蔗糖的B5培养基中摇瓶培养25 d再继代培养70 d得率最高;冬凌草毛状根提取物中含有冬凌草甲素,且能有效抑制体外培养肿瘤细胞生长。     

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     

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。     

不同理化因子对黄芩毛状根诱导的影响

目的:利用发根农杆菌1.2556诱导黄芩,得到毛状根.方法:采用共培养法诱导黄芩毛状根,研究不同外植体,不同预培养时间,不同菌液浓度,不同感染时间,乙酰丁香酮,抗生素浓度等条件对转化率的影响.结果:利用预培养2d后的茎段为转化材料,当发根农杆菌浓度在OD600值为0.5时感染10min,转化率最高.在菌液中或培养基中添加100umol/L,乙酰丁香酮可以提高黄芩毛状根的转化效率.培养基中加入250mg/L抗生素Cef能较好地抑制发根农杆菌生长.结论:用共培养法诱导出黄芩毛状根,并确定了最佳诱导条件,以提高黄芩外植体的诱导率.     

黄芪毛状根化学成分和免疫功能活性的研究

应用大规模培养技术生产的黄芪毛状根,获得10g／L的产量。与黄芪干燥根化学成分相比,黄芪毛状根中粗皂甙和可溶性多糖含量较高,黄苠甲甙含量相当,而6种异黄酮,总多糖和酸性多糖含量较步,证明两种来源的黄苠根质量相似。从免疫功能低下的小鼠免疫功能恢复的实验结果也证实了这一点。本结果显示大规模培养生产的黄芪毛状根可能成为中药材黄芪的一种新来源。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

喜树毛状根的诱导及其喜树碱含量分析

研究了不同外植体类型(包括真叶、茎段、子叶及胚轴)、胚轴年龄和不同发根农杆菌菌株(包括A4、15834、R1601、C58C1)等因素对喜树毛状根诱导频率的影响,并用PCR对诱导出的毛状根进行了分子鉴定.结果表明:(1)最佳外植体为胚轴,5~10 d是胚轴最佳诱导年龄段,最佳诱导菌株为15834.(2)PCR鉴定结果表明,发根农杆菌的rolB基因已整合到喜树毛状根基因组中.(3)对不同菌株诱导的毛状根进行HPLC检测表明,C58C1菌株诱导的毛状根的喜树碱和羟基喜树碱含量最高,分别为1.219 mg/g和0.305 mg/g.研究结果为喜树碱的药源开发提供了一条新途径,并为进一步利用基因工程技术调控喜树碱的代谢合成奠定了基础.     

蔗糖对发根农杆菌C58C1诱导烟草毛状根生长的影响北大核心CSCD

毛状根良好的生长状况是建立毛状根-AM真菌双重培养体系的关键,为优化毛状根培养基成分,确定适宜毛状根生长的蔗糖浓度,改善烟草毛状根的生长状况,该研究以发根农杆菌菌株C58C1诱导2个烟草品种NC82和Va116叶片产生毛状根,经PCR检测证实后,用含有不同蔗糖浓度的1/2MS培养基分别进行固体和液体优化培养,通过测定毛状根的分枝数、鲜重(FW)与干重(DW),研究蔗糖对2个品种烟草毛状根生长的影响。结果表明:(1)C58C1均能诱导两种烟草叶片产生毛状根,但诱导率不同,NC82(87.3%)的诱导率更高,是Va116(38.6%)的2.26倍。(2)培养基蔗糖浓度显著影响毛状根生长,因烟草品种和起始分枝数而异。(3)固体培养基优化培养NC82和Va116的毛状根,分枝数增长的抑制蔗糖浓度分别为25 g·L^(-1)和15 g·L^(-1);液体培养基优化培养分别在25 g·L^(-1)和15 g·L^(-1)时F(D)W达到最大,分别为0.541 g(0.055 g)、0.474 g(0.050 g)。(4)综合分枝数、F(D)W、毛状根生长势考虑,C58C1诱导NC82毛状根最适培养基蔗糖浓度为25 g·L^(-1),Va116毛状根为15 g·L^(-1)。该文优化了烟草毛状根培养基组成的适宜蔗糖浓度及培养方法,为后续毛状根大量扩繁奠定基础,建立了毛状根-AM真菌双重培养体系,解决了关键的寄主生长不良的问题。     

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.