桑树MaPP2C8基因克隆及其干旱胁迫下的表达

该研究基于桑树转录组测序结果及基因组数据库,采用PCR技术,克隆获得桑树2C型蛋白磷酸酶基因MaPP2C8的cDNA及其启动子序列,运用生物信息学方法对序列进行分析,并采用qRT-PCR方法检测MaPP2C8在干旱胁迫处理下的表达特性,为进一步研究MaPP2C8基因在干旱胁迫响应中的功能奠定基础。结果显示:(1)MaPP2C8基因cDNA全长为1 309 bp,开放阅读框(ORF)全长为1 053 bp,编码350个氨基酸。(2)MaPP2C8蛋白与桑科其他植物亲缘关系较近,归属于PP2Cs家族中的A亚族。(3)MaPP2C8蛋白分布于细胞中的多个位置,包括细胞质、细胞核及细胞膜等。(4)克隆获得MaPP2C8基因编码起始位点上游长度为1 612 bp启动子序列,该启动子含有3类激素相关的顺式作用元件,且与ABA相关的元件多达3个。(5)MaPP2C8基因受干旱胁迫诱导上调表达,复水处理后,其表达量显著下调。研究表明,MaPP2C8基因在桑树响应干旱胁迫过程中可能起重要作用。     

柠条锦鸡儿CkP5CS基因克隆及表达特性分析

旨在克隆柠条锦鸡儿Δ~1-吡咯啉-5-羧酸合成酶基因(P5CS)的全长cDNA序列,研究其组织表达特异性,分析CkP5CS基因在低温、高盐和PEG处理下的表达情况,以阐明CkP5CS基因在柠条锦鸡儿中的生物学功能。利用RACE技术克隆P5CS基因cDNA序列,并对其全长基因进行生物信息学分析;构建系统发育树,研究其与相似序列的同源性;利用实时荧光定量PCR方法,分析CkP5CS基因的组织表达特异性以及在低温、高盐和渗透胁迫下的表达情况。结果显示,克隆得到柠条锦鸡儿P5CS基因全长c DNA序列,命名为CkP5CS,全长2 604 bp,包括5'非翻译区(5'-UTR)102 bp,3'非翻译区(3'-UTR)363 bp,开放阅读框2 139 bp,能编码712个氨基酸,预测蛋白质分子量为76.8 kD,理论等电点为8.6,二级结构主要包括α-螺旋、延伸链和无规则卷曲。多序列比对和系统进化分析表明,柠条锦鸡儿P5CS与白刺花SdP5CS、大豆GmP5CS、豇豆VuP5CS同源性较高,分别为85.1%、84.2%和82.6%。实时荧光定量PCR结果显示,CkP5CS在柠条锦鸡儿的根、茎和叶中均有表达,没有组织特异性;同时,该基因的表达受低温、高盐和渗透胁迫的诱导。克隆得到柠条锦鸡儿CkP5CS基因,其在柠条锦鸡儿适应低温、高盐和渗透胁迫过程中发挥作用。     

野生大豆P5CS基因的克隆及对盐胁迫反应

逆境下植物大量积累脯氨酸是减轻胁迫伤害的一种自我保护机制。本研究应用同源克隆方法从NaCl处理的野生大豆中克隆获得一个脯氨酸合成酶(P5CS)基因,命名为GsP5CS。该基因核苷酸序列全长2.232 kb,含一个2148bp开放阅读框,编码715个氨基酸,包含有高等植物P5CS蛋白质的5个主要功能域,与菜豆PvP5CS1基因核苷酸序列相似性高达98.79%。Real Time PCR分析显示该基因受轻度盐胁迫诱导上调表达,根中表达高峰出现在200 mmol/L NaCl处理下,相对表达量为对照的5.83倍;叶片中表达高峰出现在300 mmol/L NaCl处理条件下,相对表达量为对照的12.78倍。并且该基因在根和叶片中的表达模式和脯氨酸含量的变化模式相同。上述结果说明,GsP5CS可能参与野生大豆脯氨酸合成。     

石蒜儿茶酚氧位甲基转移酶基因克隆与原核表达

采用同源克隆与RACE相结合的方法,首次从石蒜叶片中克隆到可能与加兰他敏生物合成相关的儿茶酚氧位甲基转移酶基因,命名为LrCOMT。核酸序列分析显示,该cDNA全长1 246bp,开放阅读框1 101bp,编码366氨基酸。氨基酸序列分析与比对发现,LrCOMT编码的氨基酸序列具有COMT蛋白的特征区域,且与鸢尾、玉米、烟草等植物COMT的同源性大于60%。将LrCOMT基因连接到原核表达载体pET-29a上,转化大肠杆菌BL21(DE3)的原核表达结果显示,重组蛋白受IPTG诱导表达,分子量大小约为40kD,与已报道的其他植物COMT大小基本一致。     

小麦TaLEC1基因的克隆及其表达特性分析

为了探讨LEC1基因在小麦(Triticum aestivum L)非生物胁迫应答中的功能,该研究通过RT-PCR结合RACE技术克隆小麦TaLEC1基因,并采用qRT-PCR方法分析了该基因在小麦不同组织以及不同处理下的表达模式,为深入研究小麦LEC1基因在干旱、高温和高盐胁迫下的响应机制奠定基础。结果表明:(1)成功克隆到小麦TaLEC1基因,该基因cDNA序列全长为1 074 bp,其中5′端非编码区23 bp,开放阅读框为741 bp,3′端非编码区310 bp,编码246个氨基酸,具有典型的CBFD_NFYB结构域。(2)实时荧光定量分析显示,TaLEC1在不同组织间表达差异显著,10 d龄幼苗的叶中表达量最高。(3)TaLEC1基因可被植物激素ABA诱导而上调表达,属于ABA依赖型的表达调控通路。(4)PEG模拟干旱胁迫处理后的0.5～1 h,TaLEC1基因呈上调表达;42℃胁迫处理过程中,TaLEC1基因呈稳定上调表达趋势,并在胁迫处理后12 h和48 h时表达急剧上调,分别为对照的52.8倍和34.5倍;NaCl胁迫处理0.5 h时TaLEC1基因迅速上调表达。研究表明,小麦TaLEC1基因参与ABA依赖的胁迫响应,推测可能在小麦耐受高温胁迫和渗透胁迫过程中发挥着重要的脱水保护功能。     

线果芥CpNSP5基因的克隆及表达分析

以该实验室前期从线果芥(Conringia planisiliqua L.)中通过mRNA差异显示技术筛选到的414bp干旱响应相关核心片段为基础,采用RACE技术对该片段进行全长克隆,序列比对分析发现,该片段与拟南芥的AtNSP5基因相似性较高,命名为CpNSP5。CpNSP5基因全长1 228bp,开放阅读框966bp,编码321个氨基酸。推测蛋白分子量为35.034 5kD,等电点为5.41。蛋白二级结构预测包含26个β-折叠,具有典型的Kelch repeat结构。系统进化分析发现,CpNSP5与白菜亲缘关系最近。实时荧光定量PCR检测发现,CpNSP5基因在20%PEG-6000和200mmol·L~(-1) NaCl胁迫下均受到不同程度诱导,说明CpNSP5基因可能与线果芥响应逆境胁迫有关。该研究结果为作物的抗旱育种提供了候选基因。     

油葵HaFAD2-2基因的克隆及表达分析

脂肪酸脱氢酶2(fatty acid desaturase,FAD2)催化油酸生成亚油酸,是植物体内生成多不饱和脂肪酸的关键酶。根据已报道的向日葵(Helianthus annuus L.)FAD2基因序列,设计引物进行RT-PCR,克隆得到油葵FAD2-2基因全长cDNA,命名为HaFAD2-2。该基因开放阅读框为1 152bp,编码383个氨基酸,相对分子质量43.96kD,等电点为8.56。对基因组进行内含子调查发现,该基因在编码区内没有内含子。多序列比对和系统进化分析发现,FAD2-2基因编码蛋白与金盏菊(Calendula officinalis)、斑鸠菊(Vernonia galamensis)等菊科植物具有较近的亲缘关系。qRT-PCR分析表明,HaFAD2-2基因在根、茎、叶、花、子叶和未成熟种子中均有表达,且以叶中的表达量最高,未成熟种子中的表达量最低;低温(5℃、15℃)胁迫处理能显著促进该基因在根中的表达,抑制其在叶中的表达;盐胁迫(300 mmol/L NaCl)处理对其表达也具有抑制作用。该研究结果可为进一步探讨HaFAD2-2基因的功能奠定基础。     

矮牵牛PhUGT74E2基因的克隆及对逆境胁迫的响应

糖基化转移酶(UGTs)能够维持植物体内的激素平衡,广泛参与植物的生长发育及逆境胁迫应答。该研究从矮牵牛(Petunia hybrida var. Mitchel diploid)中克隆了UGT74E2的同源基因PhUGT74E2及其启动子序列,并分析了序列特征和蛋白结构特点,同时采用qRT-PCR对该基因在不同组织、不同逆境胁迫下的转录水平进行了检测,以探讨矮牵牛UGT74E2基因的功能,为揭示其调控矮牵牛抗逆性的分子机制奠定基础。结果显示:(1)成功克隆获得矮牵牛UGT74E2基因全长序列,命名为PhUGT74E2。(2)PhUGT74E2基因cDNA全长1 986 bp,包含一个1 347 bp开放阅读框,编码448个氨基酸;其蛋白分子式为C_(2278)H_(3544)N_(586)O_(676)S_(18),分子量为50.53 kDa,等电点为5.18;PhUGT74E2无信号肽和跨膜域,主要定位于叶绿体;同时克隆了PhUGT74E2基因上游2 083 bp启动子序列,该序列中含有脱落酸、赤霉素、光及逆境等响应元件。(3)系统进化树分析显示,PhUGT74E2与其他物种UGT74E2起源相同,而与烟草NtUGT74E2的亲缘关系最近。(4)荧光定量PCR分析表明,PhUGT74E2基因在叶片、茎、根、叶腋和顶端5个组织中均有表达,其中叶腋中的表达量最高,而茎和根中的表达量最低;PEG6000模拟干旱处理及NaCl处理均引起了PhUGT74E2表达水平的显著上调,且随着时间的延长表达水平相应增加,说明PhUGT74E2能够参与矮牵牛对干旱及盐胁迫的响应。     

二色补血草OEE2基因的克隆和表达

从二色补血草中分离出一条含有完整开放读码框（ORF）序列的OEE2基因。该基因全长994bp,其中5’非翻译区27bp,3’非翻译区160bp,ORF全长807bp,共编码264个氨基酸,编码蛋白的分子量为28．2kDa,理论上的等电点为7．66。BlastP分析表叽二色补血草OEE2与马铃薯OEE2序列同源性最高,与喇叭水仙OEE2序列同源性最低,从9个物种的氨基酸多序列比对中可以看出,OEE2的氨基酸序列保守性较高。实时定量RT．PCR方法检测该基因对低温、NaCl和聚乙二醇（PEG）胁迫的基因表达模式的结果表明,PEG和低温能诱导OEE2基因在二色补血草叶中表达,这两种处理的OEE2基因表达量于胁迫48h后都达到高峰,而在NaCl胁迫下OEE2在二色补血草根和叶中表达都受抑制。     

文心兰铁氧还蛋白基因克隆定位及表达分析

采用RACE-PCR法,从‘小樱桃’文心兰中克隆到一个全长989bp的铁氧还蛋白基因cDNA序列,命名为OnFd(登录号KX461907)。OnFd基因开放阅读框长为465bp,预测可编码154个氨基酸;gDNA和cDNA序列比对结果显示OnFd基因不含内含子。生物信息学分析表明,OnFd具有1个典型的[2Fe-2S]结构域;同源分析显示,OnFd与玉米Fd3的相似度最高(64.29%)。蛋白亚细胞定位结果显示OnFd定位于叶绿体。实时荧光定量PCR检测发现:OnFd基因在花中表达量最高,其次是叶与根,在假鳞茎中表达量最低;接种病原菌研究显示,文心兰感染软腐病后,各个部位的OnFd基因表达量均呈上升趋势,尤其是接种部位假鳞茎在接种病原菌1d后表达量便表现出极显著上调,并且在5个感病阶段的表达量是健康植株的2.83~3.98倍。研究表明,OnFd基因可能在文心兰响应抗软腐病过程中具有重要作用。     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

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.     

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.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

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.     

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.     

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.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA