基于高通量测序鉴定细菌RNA加工位点方法的开发（英文）

简便的RNA剪切加工位点鉴定方法对于细菌核糖核酸内切酶功能和转录后水平调控机制的研究至关重要。本研究基于第二代测序技术,开发了一种能够精确鉴定RNA剪切加工位点及其剪切效率的高通量测序方法。在该方法中,首先将各种潜在RNA剪切加工的DNA片段分别克隆至报告系统进行转录,然后利用其下游特异引物进行反转录,直接构建约500 bp的双端测序cDNA文库,并在Illumina MiSeq平台对该文库进行测序。最后通过对reads 5′末端的比对定位,精确测定发生RNA剪切的位点。利用该方法,成功鉴定了来自Ruminiclostridium Cellulolyticum的cip-cel mRNA中的3个RNA剪切加工位点。与传统引物延伸和5′RACE等方法相比,该方法不仅具有更高的安全性和通量,同时还可像Northern印迹鉴定RNA的剪切效率。     

细菌非编码小RNA分子RyhB的结构与功能

RyhB是一种大小为90个核苷酸的细菌非编码小RNA分子(small noncoding RNA, sRNA).当铁缺乏时,RyhB通过下调一系列与铁的储存和利用相关蛋白的表达水平以维持体内的铁平衡,而其本身的表达则受到负调控因子Fur(ferric uptake regulator)的调节.在体内,RyhB与Hfq蛋白和核糖核酸酶E (ribonuclease E, RNase E)形成核蛋白复合物sRNP来发挥活性.sRNP通过RyhB与靶基因的互补配对序列作用于靶基因的核糖体结合位点,阻断靶mRNA的翻译,并迅速引起靶mRNA的降解.此外,RyhB还可以通过影响致病菌的生物膜形成、趋化性、耐酸性等方面的能力对细菌的致病力进行调节.本文综述了RyhB的结构、功能及作用机制方面的研究进展,并对其存在的生理意义进行了探讨.     

产肌醇酿酒酵母基因工程菌的构建

【目的】过表达酿酒酵母肌醇合成关键酶基因INO1,促进肌醇合成,构建能够分泌肌醇的基因工程菌株。【方法】构建r DNA介导的INO1基因多拷贝整合表达载体p URIH,电转化酿酒酵母Y01菌株,构建工程菌株YI2-1和YI2-2,荧光定量PCR方法分析INO1基因表达量。敲除Kan MX抗性基因,HPLC检测重组菌发酵液中肌醇含量。【结果】获得INO1基因过表达菌株YI2-1和YI2-2,YI2-1的INO1基因表达量是出发菌Y01的16.235倍。敲除Kan MX抗性基因的菌株命名为YI2-1△KP,初步检测YI2-1△KP产肌醇量为627 mg/L。【结论】r DNA介导的INO1基因多拷贝整合表达载体p URIH能够有效地过表达目的基因;过表达菌株合成的肌醇不仅能满足自身的需要,而且能够向胞外分泌,具有潜在的工业应用价值。     

里氏木霉Set2通过调控转录因子Ypr1的表达进而控制sorbicillinoids的生物合成

【目的】Sorbicillinoids是里氏木霉合成的一类重要的天然活性物质,具有抗肿瘤、抗氧化及抗病毒等多种生物活性。本研究主要是为了阐明TrSet2在里氏木霉sorbicillinoids合成调控过程中的生物学功能及其作用机制。【方法】基于生物信息分析技术鉴定里氏木霉TrSet2编码基因。采用基因敲除和过表达手段,分别构建Trset2基因敲除和过表达菌株并评估其sorbicillinoids合成能力。同时在Trset2基因敲除菌株中,过表达转录激活因子Ypr1,明确TrSet2和Ypr1之间的调控关系。【结果】Trset2基因敲除菌株完全丧失了合成sorbicillinoids的能力。相反,过表达Trset2导致sorbicillinoids合成的水平显著增加。进一步研究发现,在Trset2基因敲除菌株中过表达转录激活因子Ypr1逆转了其不能合成sorbicillinoids的表型。【结论】本研究明确了TrSet2在里氏木霉合成sorbicillinoids过程中的正向调控作用,其作用机制是通过控制转录因子Ypr1的表达水平实现的。这为基于调控机理控制里氏木霉发酵过程中sorbic...     

聚腺苷酸特异性核糖核酸酶(PARN)的功能多样性

聚腺苷酸尾的降解对于mRNA的质量控制和转录后基因调控十分重要. 在真核生物中,去腺苷酸化是mRNA降解和翻译沉默的首要限速步骤. 3′核糖核酸外切酶--聚腺苷酸特异性核糖核酸酶（poly(A)-specific ribonuclease,PARN）能够高效降解真核生物mRNA的聚腺苷酸尾. PARN不仅在降解mRNA poly(A)尾中发挥关键的作用,还参与DNA损伤、非编码RNA的加工成熟以及肿瘤等疾病过程. PARN是一种多功能酶分子,本文就PARN发现、结构、催化机制和功能多样性进行综述.     

解淀粉芽胞杆菌关键酶基因过表达对鸟苷积累的影响

【目的】研究鸟苷生物合成途径中的3个关键酶编码基因(prs,purF,guaB)过表达对解淀粉芽胞杆菌(Bacillus amyloliquefaciens)发酵生产鸟苷的影响。【方法】利用穿梭表达载体PBE43,构建含有prs、purF和guaB基因的单独表达载体和prs、purF基因的串联表达载体,将它们分别转入鸟苷生产菌B.amyloliquefaciens TA208后,通过实时定量PCR测定各工程菌株内相关基因的转录水平;通过酶活检测分析关键酶基因扩增对肌苷酸脱氢酶活性的影响;通过摇瓶发酵实验考察工程菌株与对照菌株的生长、耗糖和鸟苷积累情况。【结果】转录分析结果表明prs、purF和guaB基因过表达的同时都伴随着自身转录水平的显著上调。与此同时,prs和purF基因单独表达均轻微下调了嘌呤操纵子的转录水平,但是guaB基因的过表达并不影响嘌呤操纵子和prs基因的转录。酶活分析结果表明prs和purF基因扩增并不影响肌苷酸脱氢酶的活性,guaB基因的扩增使其活性提高了126%。摇瓶发酵实验发现prs和purF基因的单独过表达均未促进宿主菌合成鸟苷,而含guaB基因过表达载体的工程菌鸟苷产量较出发菌株提高20.7%。将prs和purF基因串联表达后,鸟苷产量提高14.4%,糖苷转化率增加6.8%。【结论】过表达guaB基因能够大幅提高鸟苷产量,而prs和purF基因只有实现协同表达才能对宿主菌积累鸟苷产生积极影响,为通过代谢工程技术提高鸟苷产量奠定了研究基础。     

苏云金芽胞杆菌dxr1基因的转录受SigH控制

【目的】萜类化合物广泛分布在生物界,是重要的生命物质。目前发现有两条萜类化合物的生物合成途径,即甲羟戊酸(MVA)途径和2-甲基-D-赤藓糖醇-4-磷酸(MEP)途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶(DXR,EC1.1.1.267)催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶,而在苏云金芽胞杆菌(Bacillusthuringiensis,Bt)中有2个基因(dxr1和dxr2)编码DXR酶。通过分析BtHD73菌株的dxr1基因的转录活性和dxr1突变体表型,明确dxr1基因的转录调控机制和功能。【方法】通过5?RACE分析dxr1的转录起始位点;β-半乳糖苷酶活性测定分析dxr1基因启动子(Pdxr1)的转录活性;采用同源重组技术分别敲除BtHD73菌株的dxr1和dxr2基因;利用总蛋白定量确定Cry1Ac蛋白产量;利用DXR检测试剂盒检测Bt菌株的DXR活性。【结果】dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基;与出发菌株HD73相比,Pdxr1在sig H突变体中的转录活性明显降低;dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响,但使DXR活性下降。【结论】Bt中dxr1基因的转录受Sig H控制,dxr1基因的缺失影响DXR的活性。     

非洲爪蛙Klf4基因新转录本的克隆及功能研究

[目的]克隆非洲爪蛙Klf4基因的新转录本,并研究其在早期胚胎发育中的功能。[方法]提取胚胎总RNA,反转录构建c DNA文库。从Xenbase数据库中获得Klf4的基因组序列,分析序列发现新的ATG位点,设计引物、PCR扩增,将扩增的目的片段插入p CS2+,插入位点为EcoRⅠ和XhoⅠ。经双酶切和测序鉴定,发现载体中插入的目的片段为编码483个氨基酸的Klf4的新转录本。体外转录获得mRNA,显微注射入胚胎,进行表型分析以及原位杂交检测。[结果]成功克隆出了Klf4基因的新转录本;过表达该转录本,表型分析发现胚胎发育异常,原位杂交检测发现Xag2和Dkk1的表达上升。[结论]成功克隆编码483个氨基酸的Klf4基因的新转录本,过表达该转录本胚胎发育异常,Xag2和Dkk1的表达被激活。     

滚环转录结合定点切割制备小RNA的技术探究

[目的]优化滚环转录及定点酶切中关键技术,提高小RNA合成量。[方法]通过在引物与模板之间设计不互补部分,形成泡状凸起,引发滚环转录;使用天然DNAzyme 10-23定点剪切转录产物;另外改变RNase H酶切中Aid-DNA修饰核酸数目及位置,指导RNase H切割;DNaseⅠ降解模板DNA后,不经高温失活,直接进入RNase H酶切体系。[结果]由凸起结构引发转录,转录效率提高约5倍;RNase H在仅有3个修饰核酸间隔分布的Aid-DNA-3b辅助下,可高效定点剪切,而DNAzyme 10-23无法充分切割滚环转录产物;未失活的DNaseⅠ对Aid-DNA-3b的降解仅占8.7%,可直接进入RNase H酶切体系。[结论]引入凸起结构可提高转录效率约5倍;DNaseⅠ可直接进入酶切体系,随后使用Aid-DNA-3b介导酶切,产量可提高1.4倍。     

过表达TatAdCd转位酶对枯草芽孢杆菌脂肪酶分泌的影响

【目的】研究过表达枯草芽孢杆菌Tat运输途径的Tat Ad Cd转位酶对促进脂肪酶分泌的影响。【方法】用cdd基因的串联启动子和前导区,替换tat AD-CD操纵子的启动子和前导区,并在染色体sac B基因位点整合表达;采用q RT-PCR方法表征tat AD-CD操纵子的表达水平;用脂肪酶表达质粒p HP13L转化Tat Ad Cd转位酶过表达菌株,构建产脂肪酶重组菌。通过测定脂肪酶活性,以及聚丙烯酰胺凝胶电泳,考察Tat Ad Cd转位酶过表达对脂肪酶分泌的影响。【结果】tat AD-CD操纵子被过表达,其胞内m RNA相对水平提高了185倍。Tat Ad Cd转位酶的过表达,使脂肪酶发酵单位提高了40%。【结论】使用cdd基因的串联启动子和前导区,能够有效地过表达目的基因;枯草芽孢杆菌脂肪酶可以同时经由Sec途径和Tat途径分泌;过表达Tat Ad Cd转位酶,能够显著提高脂肪酶的分泌量。     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.