柑橘黄龙病罹病植株显症差异组织内生细菌群落结构分析

【目的】分离鉴定同株罹病柑橘黄龙病植株不同显症状况组织的内生细菌,寻找与黄龙病菌[‘Candidatus Liberibacter asiaticus’(Ca.Las)]相互作用的优势菌株。【方法】利用基于16S rDNA的PCRDGGE(Denaturing gradient gel electrophoresis)分析同一柑橘黄龙病罹病植株的显症和未显症组织内生细菌多样性,并用定量PCR方法,对果、枝、叶3种组织黄龙病菌、优势菌株及细菌总数进行检测。【结果】结果显示显症和无症组织所带黄龙病菌差异很大,显症部位病菌量明显高于无症部位。分析显症和无症组织内生细菌DGGE图谱显示,同一组织内生菌群结构基本相同;对图谱中17条明显条带回收克隆测序,发现其中8个条带均属于沙雷氏菌属(Serratia),占总条带数的47.06%。序列分析显示这8条序列为粘质沙雷氏菌(Serratia marcescens)不同的菌株(序列相似性为99.63%)。定量分析各差异显症部位单位组织内的粘质沙雷氏菌和细菌总数,发现相同部位的总细菌量差异不显著,但粘质沙雷氏菌与黄龙病菌的量呈负相关。【结论】柑橘黄龙病病株中,各部位所带病菌量不均匀,是否显症与组织内柑橘黄龙病菌的量呈正相关,内生菌群总量与显症无相关性,但粘质沙雷氏菌与黄龙病菌的量呈负相关。粘质沙雷氏菌与黄龙病菌在韧皮部细胞内增殖过程中的相互作用值得深入研究。     

副溶血性弧菌重复序列-PCR分型研究

利用基因外重复回文序列-PCR(REP-PCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)技术,对副溶血性弧菌进行了分子分型研究和亲缘关系的探讨,并使用Hunter和Gaston方法计算分辨力指数.结果显示40株副溶血性弧茵分离株均可扩增产生可重复的DNA指纹图谱,并且不同菌株基因组DNA的扩增条带具有多态性.根据SPSS10.0软件得出的树状图结果,REP-PCR可以把40株茵分为21个型,分辨力指数可达到0.953,优势菌型为G1型;ERIC-PCR可将40株菌分成4个型,分辨力指数为0.5.研究显示重复序列-PCR方法可以用于该菌分型分析,REP-PCR具有较好的分型能力.在两种PCR的DNA指纹图谱中,血清型O1群与O3群主条带均非常相似,表明它们之间亲缘关系密切.     

瓜类枯萎病菌遗传多样性和亲缘关系的SRAP分析

尖孢镰刀菌可造成不同瓜类的枯萎病.为明确不同寄主、不同地区的瓜类枯萎病菌菌株间的遗传多样性及亲缘关系,采用相关序列扩增多态性(SRAP)分子标记技术,对来源于不同地区、不同寄主的95株尖孢镰刀菌的基因组DNA进行多态性扩增.以筛选出的19对引物共扩增出238条带,多态性比率为100%,平均每对引物扩增出12.5个位点和12.5个多态性位点;尖孢镰刀菌苦瓜专化型共扩增出166条带,其中145条为多态性条带,多态性比率为87.4%,平均每对引物扩增出8.7个位点和7.7个多态性位点,说明尖孢镰刀菌的遗传变异较为广泛.瓜类枯萎病菌株间的遗传相似系数范围为0.68～0.99,样品间的平均Nei遗传多样性指数和Shannon指数分别为0.2390和0.3718.在遗传相似系数为0.74时,可将供试的95株尖孢镰刀菌划分为苦瓜、黄瓜、西瓜、甜瓜4个专化群.在SRAP聚类树中,同一寄主的尖孢镰刀菌聚在一个分支上,其中尖孢镰刀菌苦瓜专化型菌株间的遗传相似系数范围为0.78～0.99,Nei遗传多样性指数为0.1811,平均Shannon指数为0.2750,表明尖孢镰刀菌苦瓜专化型的遗传变异较大,且菌株的聚群与地理来源存在相关性.     

地衣内生菌Elaphocordyceps sp.的代谢产物

【目的】通过对猫耳衣内生菌Elaphocordyceps sp.代谢产物的研究,以期获得结构新颖、抗菌活性强的化合物。【方法】采用PCR扩增和测定nrDNA的ITS(Internal transcribed spacer)序列,在GenBank数据库中比对,并采用构建系统发育树的方法,确定该菌株的分类地位。采用硅胶柱色谱、凝胶柱色谱和高效液相色谱方法进行成分分离。根据理化性质和波谱数据进行结构鉴定。采用倍比稀释法进行抑菌活性测定。【结果】鉴定出供试菌株为地衣内生菌Elaphocordyceps sp.。从Elaphocordyceps sp.的发酵液中分离得到3个化合物,结构鉴定为麦角甾醇、4,8-Dihydroxy-1-tetralone和De-O-methyldiaporthin。化合物1–3表现弱的抑制白色念珠菌活性。化合物3具有非常强的植物毒活性(叶片敏感度:4 nmol)。化合物3在地衣内生菌Elaphocordyceps sp.发酵液中的得率为0.75 mg/L(3.2?103 nmol),远远高于其对叶片敏感度4 nmol。【结论】由猫耳衣内生菌Elaphocordyceps sp.产生的De-O-methyldiaporthin可作为一种微生物源除草剂。     

肺囊康定产生菌Glarea lozoyensis线粒体基因组序列的再分析（英文）

【目的】Glarea lozoyensis是抗真菌药物卡泊芬净的产生菌,其突变菌株ATCC 74030的线粒体基因组已被报道。我们此前的研究发现诱变剂能引起该菌某些细胞核基因的突变,但诱变剂是否也能引起线粒体DNA序列的改变并不清楚。【方法】组装野生型菌株ATCC 20868的线粒体基因组,并与发表的突变型菌株ATCC 74030的线粒体基因组进行比较。通过PCR验证野生和突变菌株线粒体基因组间表现差异之处,并利用正确的线粒体基因组序列进行新的分析。【结果】我们成功组装出野生型菌株ATCC20868的线粒体基因组,通过比较其与发表的ATCC 74030的线粒体基因组序列,发现存在6处单核苷酸变异位点和2处具有长度差异的区域。然而,随后的PCR验证和序列比较并没有发现2个菌株间存在这些差异。最初观察到的碱基差异是因为发表的ATCC 74030线粒体基因组存在序列错误。有趣的是,在Glarea lozoyensis的线粒体基因组中,我们发现存在3个具有内含子的t RNA基因和1个rnp B基因。同时,该菌线粒体基因组中存在多种重复序列,在其线粒体和细胞核基因组间也存在明显的DNA片段重复事件。【结论】诱变剂没有引起G.lozoyensis线粒体DNA的任何改变;发表的ATCC 74030的线粒体基因组存在序列错误。我们报道G.lozoyensis正确的线粒体基因组序列,并且发现该菌线粒体和细胞核基因组间频繁的基因交流。     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

副溶血性弧菌重复序列-PCR分型研究

利用基因外重复回文序列-PCR(REP-PCR)和肠细菌基因间共有重复序列-PCR(ERIC-PCR)技术, 对副溶血性弧菌进行了分子分型研究和亲缘关系的探讨, 并使用Hunter和Gaston方法计算分辨力指数。结果显示40株副溶血性弧菌分离株均可扩增产生可重复的DNA指纹图谱, 并且不同菌株基因组DNA的扩增条带具有多态性。根据SPSS10.0软件得出的树状图结果, REP-PCR可以把40株菌分为21个型, 分辨力指数可达到0.953, 优势菌型为G1型; ERIC-PCR可将40株菌分成4个型, 分辨力指数为0.5。研究显示重复序列-PCR方法可以用于该菌分型分析, REP-PCR具有较好的分型能力。在两种PCR的DNA指纹图谱中, 血清型O1群与O3群主条带均非常相似, 表明它们之间亲缘关系密切。     

引起芭蕉芋细菌性软腐病的玉米迪基氏菌(Dickeya zeae) CE1的全基因组序列及比较基因组学分析

【背景】玉米迪基氏菌(Dickeya zeae)可引起香蕉、水稻等重要作物的细菌性软腐病,并造成巨大的损失。芭蕉芋抗性较好且与病虫害相关的报道很少,本研究团队首次报道了由D. zeae CE1引起的芭蕉芋细菌性软腐病。【目的】揭示CE1菌株的全基因组序列,并与同样来源于广东香蕉和水稻的D. zeae菌株作比较基因组学分析,初步探讨D. zeae种内不同病原细菌在与寄主互作过程中可能存在的遗传分化机制。【方法】采用三代测序结合二代测序对CE1菌株进行完整基因组测序,利用比较基因组学方法分析该菌株与香蕉和水稻菌株的进化关系和基因组特征差异。【结果】细菌基因组测序表明,CE1菌株的完整基因组大小为4 714 731 bp,注释预测到4 052个编码基因。与芭蕉芋和香蕉两个寄主亲缘关系类似,基因组比较分析发现来自芭蕉芋和香蕉的病菌菌株亲缘关系较近,它们在遗传进化上明显不同于水稻菌株。基因家族分析表明,编码重要致病因子如细菌分泌系统、鞭毛蛋白、胞外多糖、规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)等的基因簇在不同寄主间未表现明显分化。通过进一步分析发现,有80个基因是芭蕉芋和香蕉菌株所特有的,42个基因则是水稻菌株所特有的。在芭蕉芋和香蕉菌株的特异基因中,功能预测发现有2个基因簇分别与脂肪酸合成酶和群体感应相关;然而较多的水稻菌株特异基因则与碳水化合物转运和代谢相关,另外有一个水稻菌株特异基因簇存在于CRISPR的邻接位点。【结论】比较基因组学分析确定了芭蕉芋菌株和香蕉菌株、水稻菌株间的遗传亲缘关系,并发现了数个可能与不同类型寄主互作相关的基因位点,为D. zeae病原细菌侵染与香蕉、水稻等重要作物亲缘关系较为接近的不同作物提出风险预警。     

赣南柑橘木虱体内黄龙病菌psy62株系原噬菌体类型多样性的研究

为探明柑橘木虱体内黄龙病菌psy62株系中原噬菌体遗传多样性及与柑橘株系的遗传差异,本研究将利用3对引物在psy62基因组中2个噬菌体FP1和FP2的同源序列区域内对赣南地区阳性样品进行PCR扩增和测序。结果显示,赣南地区阳性样品在此基因区域内有28头柑橘木虱检测到原噬菌体,共有5种类型,分别为A,A2,B,C,C1。其中A,B的检测率相对较高;C1检测率最低,为7.1%。由此可见,在此基因位点不同地理位置的柑橘木虱原噬菌体种群有较大的遗传差异,且与美国佛罗里达的"Ca.L.asiaticus"株系,中国柑橘叶片"Ca.L.asiaticus"株系均有显著的遗传差异,这有助于进一步研究分析原噬菌体的种类差异对柑橘木虱传毒机制的影响。     

应用PCR技术对DMD患者进行缺失检测与产前基因诊断

运用聚合酶链式反应(polymerasechainreaction,PCR)技术对3个Duchenne型肌营养不良症(DMD)家系中的患者进行dystrophin基因内9个外显子缺失检测,在2个家系中检测到外显子45、48、51缺失,同时运用PCR技术扩增位于dystrophin基因内内含子短串联重复序列,对非缺失型DMD家系进行了产前诊断,胎儿为正常女性.dystrophin基因外显子缺失检测方法快速、敏感、准确,可在临床推广中应用;短串联重复序列(STR)多态性分析方法可用于DMD家系的产前基因诊断和携带者检出.     

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.