苇状羊茅内生真菌与多年生黑麦草内生真菌实时荧光PCR检测研究

苇状羊茅内生真菌Neotyphodium coenophialum和多年生黑麦草内生真菌N．lolii对美国、新西兰等国家的畜牧业曾经造成过巨大损失。以N．coenophialum和N．lolii及其近似种N．huerfanum、N．chisosum、N．aotearoae、N．sp．共6种18个菌株,以及苇状羊茅和多年生黑麦草8个品种种子为供试材料,根据Tub-2基因设计了通用Taqman探针及引物,根据NC25基因设计了N．coenophialum和N．lolii的特异Taqman探针及共用引物,通过通用探针的单色荧光PCR和特异探针的双色荧光PCR,建立了N．coenophialum和N．lolii的菌丝及单粒种子稳定可靠、特异性强的荧光PCR检测方法,检测灵敏度达到单粒种子,使检测时间由至少一个月缩短至7～8个小时。     

Neotyphodium coenophialum和N.lolii的PCR检测*

以N·coenophialum和N·lolii及其近似种N·huerfanum、N·chisosum、N·aotearoae、N·sp·共6个种18个菌株及苇状羊茅和多年生黑麦草8个品种的供试种子,通过对Tub-2基因引物IS1~IS3和NC25基因引物F1~R1进行扩增,设计了种子中Neotyphodium属内生真菌套式扩增的通用引物Tub-2-F~Tub-2-R,设计了N·coenophialum和N·lolii的特异引物F3~R3     

高羊茅和多年生黑麦草内生真菌的分子检测

根据GenBank上报道的内生真菌Neotyphodium coenophialum和N.lolii的Nc25基因序列,设计了2对特异性引物FM1/R1和F3/R652,建立了一套适合于从高羊茅和多年生黑麦草种子中检测内生真菌N.coenophialum和N.lolii的常规PCR和巢式PCR方法。     

利用RT-LAMP可视化检测技术检测H1亚型禽流感病毒及N1、N2亚型的分型

为直接根据颜色变化进行可视化检测H1亚型、N1亚型、N2亚型禽流感病毒(AIV),根据环介导等温扩增技术(LAMP),建立针对H1亚型AIV及特异性鉴定N1、N2亚型的逆转录环介导等温扩增(RT-LAMP)检测方法。根据GenBank中的AIV基因序列,设计了三套分别针对H1亚型AIV-HA基因及N1、N2亚型AIV-NA基因的特异性简并引物,并优化反应条件和体系。结果表明建立的检测方法对其它亚型AIV及禽呼吸道病原体无交叉扩增反应并能特异性地检测N1、N2亚型AIV,灵敏度优于传统的RT-PCR方法。整个反应在常规水浴中50min就可完成,反应结束后不需打开反应管盖,可根据反应液的颜色变化对结果直接进行判定。120份临床样品用建立的RT-LAMP方法检测到14份H1N1亚型AIV、8份H1N2亚型AIV,结果与病毒分离结果相符。本研究建立的三种RT-LAMP可视化检测技术特异、灵敏、快速、操作和结果判定简便,适合在基层进行H1亚型AIV的快速检测及N1、N2亚型AIV的分型。     

检测肠道细菌Feacalibacterium prausnitzii的三对PCR引物的特异性比较

【目的】比较3对基于16S rRNA基因、用于检测人肠道中重要细菌Feacalibacterium prausnitzii的引物(FPR-1/FPR-2、FPR-2F/Fprau645R和Fprau223F/Fprau420R)的特异性。【方法】用Clustal X比对每个引物与F.prausnitzii和其他细菌的16S rRNA基因的序列。在Ribosomal Database Project(RDP)数据库中使用Probe Match工具比较每个引物匹配的Faecalibacterium spp.序列数目。利用本课题组建立的中国人粪便菌群的16S rRNA基因全长文库的7255个克隆序列,用Simulated PCR(SPCR)预测每对引物检测到的F.prausnitzii和其他细菌的克隆数;用3对引物分别对代表克隆进行PCR扩增。用3对引物分别对14个健康人的粪便样品进行实时定量PCR。【结果】引物Fprau645R的3端最后一个碱基与非F.prausnitzii序列的错配度高于其它引物,它在RDP中匹配的Faecalibacterium spp.序列数占其匹配的细菌序列数的百分比(97.6%)显著高于其他引物。SPCR预测,3对引物检测到的F.prausnitzii克隆数均为1171左右;在检测到的非Faecalibacterium spp.克隆中,FPR-2F/Fprau645R主要是Subdoligranulum spp.,而FPR-1/FPR-2和Fprau223F/Fprau420R还有Oscillibacter spp.、Ruminococcus spp.和unclassified Ruminococcaceae等。真实PCR与SPCR的结果吻合。实时定量PCR中,FPR-1/FPR-2和Fprau223F/Fprau420R检测到的细菌数量高于FPR-2F/Fprau645R。【结论】3对引物能检测到F.prausnitzii和Subdoligranulum spp.,FPR-2F/Fprau645R的特异性优于FPR-1/FPR-2和Fprau223F/Fprau420R。     

甲型H1N1流感病毒HA基因RT-LAMP检测方法的建立与初步应用

目的:建立反转录环介导等温扩增技术(RT-LAMP)检测甲型H1N1流感病毒HA基因的方法,并用于检测临床样本.方法:通过在线软件Primer Explorer V4设计H1N1 HA基因的RT-LAMP引物,建立RT-LAMP检测方法,并评价其灵敏度和特异度.结果:与传统RT-PCR方法相比,RT-LAMP检测方法具备更高的灵敏度,达到10个拷贝,并且具有良好的特异性;在76份呼吸道感染儿童的咽拭子标本中检测到2份阳性,与RT-PCR方法检测结果相同.结论:建立的H1N1 RT-LAMP检测方法灵敏特异,简单快速,具备H1N1现场检测应用前景.     

季节性流感病毒H1N1实时荧光定量PCR检测方法的建立

目的建立一种快速定量检测季节性流感病毒H1N1核酸的实时荧光定量PCR检测方法及试剂盒。方法选择季节性流感病毒H1N1的保守基因NP基因作为检测靶目标,应用Clustal W软件进行序列同源性比对分析,筛选出季节性流感病毒H1N1特异性的保守序列作为引物候选区域,然后应用Primer Express及PrimerPremier 5.0软件包对候选引物进行进一步配对及筛选,得到最优特异性检测引物。同时,由病毒全长cDNA扩增出NP基因,琼脂糖凝胶电泳检测NP基因的扩增情况并对目的条带进行切胶回收及纯化,对回收后的NP全长基因进行核酸浓度测定,并换算成拷贝数,作为定量标准品。结果应用ABI公司的Power SYBR Green PCR MasterMix及StepOne实时荧光定量PCR仪,该检测系统灵敏度可达102 copies/μL,不同梯度标准品间线性关系（R2）达0.999,斜率为-0.3433,扩增效率为95.572%,所有标准品均在83.2℃出现尖且窄的特异性熔解峰。结论利用该检测系统可以快速定量检测季节性流感病毒H1N1,灵敏度高,可用作基础及临床实验室对季节性流感病毒H1N1感染的辅助诊断方法和临床效果的监测手段,对实验操作者要求相对较低,具有实际的应用价值。     

基于16S rDNA基因的谷斑皮蠹PCR检测技术

【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL~(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL~(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。     

猪FGL2基因cDNA末端序列检测及结构分析

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α-32P dCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapid amplification of cDNA end,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage 2 聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。     

Morphological,cytological and molecular characterization of a novel symmetric somatic hybrid between N. tabacum and N. glauca

Abstract

By using the protoplast fusion technique, we have obtained 44 regenerated plants, phenotypically different and distinct from their parents, among which we have identified a fertile symmetric somatic hybrid, designated as TG-32, between N. tabacum var. Gexin No.1 and N. glauca. The morphology, fertility, chromosome number and nuclear constitution of the somatic hybrid have been studied in detail. Unlike other asymmetric interspecific somatic hybrids, the chromosome number of the symmetric somatic hybrid is 72, equal to the sum of chromosomes of both parents. The TG-32 plant has flowers similar to those of N. tabacum, but with petals similar to those of N. glauca. Interestingly the offspring of TG-32 vary in seed production ability with temperature, and produce more seeds under a relatively low temperature. Two SCAR markers were used to evaluate genetic variability and structure. The hybrid amplified the expected fragment, but the parents showed only one of two markers. This experimental result supports the hypothesis of the co-existence of two parental genomes in the somatic hybrid.     

假地枫皮的订正

作者查阅了全国42个标本馆所收藏的403号假地枫皮Illicium jiadifengpi B.N.Chang标本, 确认该种植物为中国特有, 分布于安徽、浙江、江西、福建、湖北、湖南、广东和广西。将百山祖八角I.jiadifengpi B.N.Changvar.baishanense B.N.Chang et S.H.Ou归并到假地枫皮;将闽皖八角I.minwanense B.N.Changet S.D.Zheng作为假地枫皮的一个变型--紫花八角I.jiadifengpi B.N.Changf.minwanense(B.N.ChangetS.D.Zhang)Q.Lin, 将武夷八角I.wuyishanum Q.Lin归并到紫花八角中。     

A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata

 Cytoplasmic somatic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Nicotiana alata were constructed. A total of 33 green regenerants were obtained after fusion of protoplasts from a tobacco cytoplasmic chlorophyll-deficient mutant and gamma irradiation-inactivated leaf protoplasts of N. alata. Twenty nine of them were male sterile and displayed an altered stamen morphology (formation of petaloid and stigmoid structures instead of stamens). Southern-blot analyses of eight CMS plants using N. alata-specific nuclear repetitive DNA and cpDNA probes revealed that they contained nuclear genetic material of N. tabacum and chloroplasts from N. alata. Restriction-enzyme analysis of mitochondrial DNAs of the cybrids in question showed different patterns consisting of an incomplete mix of mtDNA fragments from both parents, as well as new fragments. Southern-blot analysis of mtDNAs with a sunflower atpA probe gave the same recombinant hybridization pattern for all analyzed cybrids, indicating that high-frequency specific recombination occurs in the atpA region. Analysis of the progeny from three successive backcrosses of the studied cybrids with N. tabacum demonstrated a strict cytoplasmic inheritance of the male-sterile phenotype. Received: 10 May 1997 / Accepted: 31 March 1998     

70株淋球菌的质粒及对青霉素敏感性的检测

本文检测的70株淋球菌含三种质粒,质粒总检出率为95.7％(67株)。均含2.6Md者有67株,占94.29％,可能是隐蔽质粒。含3.2Md及23.1Md者20株均对青霉素耐药,故含此两种质粒的淋球菌有可能成为哈市耐青霉素的流行株。提取2.6Md质粒有希望制备诊断的探针。     

Taxonomic studies on the diatom genus Navicula Bory. Navicula grevillii (CA.Ag.) Heiberg and N. comoides (Dillwyn) H. & M. Peragallo

Two closely related species of the diatom genus Navicula were investigated using light and electron microscopy. A hitherto unrecorded structure associated with the raphe of N. delognei Van Heurck is described.
Examination of type material revealed that the nomenclatural types of N. grevillii (CA.Ag.) Heiberg and N. comoides (Dillwyn) H. & M. Peragallo do not agree with the present understanding of these species, therefore alternative names were sought. All the synonyms of each species were traced and a table of synonymy is supplied. The names N. delognei and N. pseudocomoides are proposed as replacements for N. grevillii and N. comoides respectively and types are cited.     

淋球菌的营养分型及质粒的生态分布

淋球菌营养型及质粒类型是其重要的生物表型。我们对９３￣９４年度在长春地区分离的９８株淋球菌,进行了营养型分布,质粒微生态分布的实验研究,结果,属于原养型（Ｗｒ）３８株,占３８．７％;ＡＨＵ＾－型３４株,占３４．７％;Ｐｒｏ＾－型１７株,占１７．４％;Ｓｅｒ＾－型６株,占６．１２％;Ｉｌｅ＾－型３株,占３．１％,未见ＰＡＶ＾－型。所有菌株中,共检出四种类型的质粒,其分子量分别为２．６Ｍｄ,４．５Ｍｄ     

山楂和山楂叶的氨基酸分析

本文用835-50型氨基酸自动分析仪测定了山楂和山楂叶中17种氨基酸的含量。结果表明,山楂叶中氨基酸的总含量高达10.3％,山楂中约含2.4％。     

Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.     

脲酶／硝化抑制剂对土壤有效态氮、微生物量氮和小麦氮吸收的影响

研究了脲酶抑制剂(NBPT)、硝化抑制剂(DCD)及二者组合在草甸棕壤上施用对尿素态N转化及土壤总有效态N、微生物量N的影响．结果表明,尿素配施NBPT、DCD及抑制剂组合能够增加尿素水解后土壤NH4^+含量2％-53％。显著降低了氧化态N的浓度,抑制了土壤中铵态N的氧化,增加土壤总有效N34％-44％,小麦吸N量增加0．26％-6．79％。其中以脲酶抑制剂与硝化抑制剂组合的效果最明显．抑制剂施用增加了微生物在小麦生长初期对有效态N固持,有利于后期土壤有效态N的矿化．     

N. I. Vavilov-the man and his work

A brief sketch is presented of Vavilov's career, his main contributions to science and personal qualities. The imporatnce of his work in genetics, agro-ecology, plant breeding, the experimental taxonomy of crop plants and genetic resources conservation is also emphasized.