蝴蝶兰花瓣瞬时转化体系建立

蝴蝶兰花器官中基因功能的研究受遗传转化效率低和遗传转化周期长的制约,而花瓣瞬时表达体系是一种快速分析基因功能的有效手段。该研究以蝴蝶兰‘大辣椒’花瓣和萼片为实验材料,通过农杆菌介导的瞬时转化方法,分析了侵染的菌液浓度、侵染时间、乙酰丁香酮浓度和共培养时间等4个因素对β-葡糖醛酸酶(GUS)报告基因表达效率的影响,以探寻其瞬时表达的最佳条件;并将查尔酮合成酶(chalcone synthase,CHS)基因RNAi干扰载体瞬时转化蝴蝶兰花瓣,共培养3d后观察转化材料中花色表型以及色素的变化,并利用半定量RT-PCR来检测CHS基因转录水平的表达。结果表明:(1)农杆菌菌液OD600为0.6、侵染时间60s,在重悬液中添加150μmol/L乙酰丁香酮,共培养3d,GUS瞬时表达率最高(85.01%)。(2)转基因蝴蝶兰花瓣颜色明显变淡,色素含量降低。(3)半定量PCR检测表明,CHS基因的转录活性相比于对照组显著降低。该实验成功的在蝴蝶兰花器官中建立了一种快速基因功能验证方法,为后期蝴蝶兰基因功能研究和育种工作提供技术支持。     

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％。     

影响花椰菜农杆菌介导转化因素的研究

以花椰菜赛雪的带柄子叶为外植体,以MS为基本培养基,GUS基因为报告基因,分析了遗传转化过程中的影响因子,如预培养时间、农杆菌菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度、延迟筛选时间等对外植体瞬间表达和稳定表达的影响。结果显示,以花椰菜的带柄子叶为外植体,预培养2d,农杆菌菌液为OD6000.3～0.4,侵染8min,共培养2d,乙酰丁香酮浓度为100μmol/L,延迟筛选7d,卡那霉素筛选压为5mg/L为最优的遗传转化方案,转化率最高可达35.7%。另外,GUS瞬间表达率和转化率并不存在绝对的相关性,但瞬间表达分析仍然可以作为外源基因进入受体细胞的指示。花椰菜农杆菌介导转化方案的优化研究为芸薹属蔬菜高效遗传转化提供了技术保障,有利于芸薹属蔬菜遗传育种与种质创新研究。     

根癌农杆菌介导转化谷子的影响因素

利用GUS报告基因,建立了农杆菌介导的谷子(Setaria italica)遗传转化体系,并研究了多种影响因素对转化效率的影响,包括受体基因型、外植体类型、菌液浓度、培养基中乙酰丁香酮的浓度、侵染时间和共培养时间.确定了最优的转化条件为:以谷子幼穗诱导的愈伤组织为外植体,用低浓度的农杆菌菌液侵染30～40min,然后在含有0.1mmol/L乙酰丁香酮的LS培养基上共培养2d.     

EuFPS基因表达载体构建及对杜仲遗传转化的研究

本实验用EcoRⅠ和BamHⅠ双酶切植物表达载体pSH737和含有目的基因的pUC-FPS,定向连接得到重组质粒pSH-FPS,将其导入农杆菌EHA105.采用农杆菌介导法对杜仲进行遗传转化,研究了卡那霉素(kanamycin,Km)浓度、预培养时间、菌液浓度及侵染时间、乙酰丁香酮(acetosyringone,AS)浓度、共培养时间等对杜仲遗传转化效率的影响.结果表明,选择无菌苗苗龄15 d的杜仲下胚轴,卡那霉素浓度50mg/L,农杆菌浓度OD600值0.3-0.6,侵染时间8 min,侵染时菌体重悬液中添加50 μmol/L乙酰丁香酮,共培养时间3 d,抗性芽的获得率最高.对再生植株进行GUS检测发现有45%的植株呈阳性.     

影响农杆菌介导狗牙根遗传转化的因素

农杆菌LBA4404/pCAMBIA1301介导转化狗牙根的体系中,遗传转化的最佳优化条件是:胚性愈伤组织预培养10 d,农杆菌菌液浓度为OD600 0.5～0.8,共培养时间为2d.共培养基中添加100μmol·L-1乙酰丁香酮能有效地提高植物瞬时表达率.侵染处理方法中滤纸滴加法比浸泡法效果更好.黑暗条件下的瞬时表达率比12 h光照/12 h黑暗培养条件下的高.在最佳优化条件下狗牙根的GUS瞬时表达率达到36.36%.     

大豆胚尖遗传转化体系优化及抗逆基因GmPK转化研究

采用GUS基因瞬时表达检测方法,通过正交试验以AS浓度、侵染菌液OD值、侵染时间、共培养时间和恢复培养时间5个因素在4个水平上进行分析,优化了农杆菌介导的大豆胚尖遗传转化体系,并在此基础上进行了抗逆基因GmPK的遗传转化。结果表明,采用共培养培养基中添加100μmol/L AS、侵染菌液OD600值0.9、侵染15h、共培养5d和恢复培养3d的转化条件最佳,GUS阳性率达74.59%,经PCR及RT-PCR进一步验证获得了转基因阳性植株。利用优化的最佳条件进行抗逆基因GmPK的转化,炼苗移栽成活的再生植株经PCR及PCR-Southern blotting验证,初步证明外源基因已经整合至大豆基因组,转化率为0.6%。     

根癌农杆菌介导的金发草遗传转化条件的优化

金发草(Pogonatherum paniceum)是一种多年生岩生草本植物,在生态恢复和景观建设中起着重要的作用。利用根癌农杆菌介导转化金发草胚性愈伤组织,通过GUS(β-葡萄糖苷酸酶)瞬时表达率研究菌液浓度、浸染时间、乙酰丁香酮(AS)浓度、葡萄糖浓度、共培养时间等因素对金发草转化的影响,并利用确定的最佳条件将GUS基因转入金发草,获得稳定表达转化植株。结果表明:菌液浓度(OD₆₀₀)为0.6,浸染时间为10min,添加20mg/L的乙酰丁香酮(AS)和10g/L的葡萄糖,共培养时间5d为最佳条件,GUS瞬时表达率最高。经过抗性筛选后最终获得阳性转基因植株频率为57%。再生植株经GUS染色和PCR检测证明,GUS基因已成功整合到金发草基因组中。此转化体系的建立为金发草的遗传改良及相关功能基因的研究奠定了基础。     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。     

农杆菌介导佛手遗传转化主要影响因素的研究

采用根癌农杆菌介导的佛手叶盘转化法,在建立转海藻糖合酶基因(TPS)佛手体系过程中,对影响农杆菌转化频率的各种因素进行了研究。结果表明,佛手叶盘需在黑暗条件下MT培养基上预培养2-3d,与农杆菌共培养3d较合适;农杆菌菌液浓度OD600约为0.6-0.8,感染时间20min;抑制农杆菌生长的抗生素浓度以头孢霉素(Cef)250mgL-1和羧苄青霉素(Cb)250mgL-1且延迟筛选时间4d最好;共培养基中添加100μmol/L乙酰丁香酮(AS)和400mgL-1半胱氨酸(L-Cys)对佛手遗传转化有明显的促进作用。经GUS报告基因和PCR技术检测,初步证实TPS基因已整合到佛手基因组中,且GUS报告基因瞬时表达率为5.9%。     

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.     

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     

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.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV