鸭儿芹肉桂酸4-羟化酶基因的克隆与不同温度下的表达分析

为研究鸭儿芹木质素合成相关基因,以贵州省野生鸭儿芹为研究对象,克隆到1条1 631bp长的肉桂酸4-羟化酶(C4H)基因(CjC4 H),并以鸭儿芹生长过程中主要面临的4种不同温度(10℃、18℃、30℃和38℃)处理0、0.5、1、2、4、8、12和24h,考察鸭儿芹木质素合成基因CjC4 H的表达情况,探讨鸭儿芹木质素合成基因受温度的调控关系,为鸭儿芹适宜生长温度的选择提供参考。开放阅读框(ORF)分析显示,CjC4 H基因的开放阅读框(ORF)长度为1 521bp,可编码506个氨基酸,蛋白分子质量为58.13kD,等电点为9.38。进化树分析表明,同科植物之间C4H进化上相对保守。实时定量PCR检测鸭儿芹中CjC4 H基因与温度的响应表达,结果表明38℃高温处理4h时,CjC4 H基因表达量远高于其他温度处理;而10℃和18℃相对低温处理,CjC4 H基因表达在24h时明显提高。     

香鳞毛蕨DfDREB基因克隆与表达模式分析

DREB(dehydration-responsive element-binding)转录因子是响应非生物胁迫反应的主要调节因子,为探索DREB在多年生草本药用植物香鳞毛蕨[Dryopteris fragrans(L.) Schott]抗逆过程中的功能,该研究克隆了DfDREB基因并进行生物信息学分析,采用qRT-PCR方法分析了DfDREB基因在不同激素及干旱、NaCl、高温和低温等逆境胁迫处理下的表达模式。结果表明:(1)成功获得DfDREB基因全长1 203 bp,其编码401个氨基酸,相对分子质量为43.66 kD,等电点为6.13,是亲水性非分泌蛋白,该蛋白具有AP2保守结构域,属于AP2家族。(2)实时荧光定量PCR分析表明,DfDREB基因在香鳞毛蕨根、叶柄和叶中均有表达,其中在叶中表达量最高,根中表达量最低;在水杨酸(SA)、茉莉酸甲酯(MeJA)和乙烯利(ETH)处理时,DfDREB基因表达上调,并都在1 h时表达量达到峰值;在脱落酸(ABA)处理时,DfDREB基因的相对表达量仅在12 h和24 h时呈上调表达,其余时间为下调表达;在干旱、NaCl和高温处理时,DfDREB基因表达均上调;低温处理0.5 h和12 h时DfDREB基因表达显著上调,但在低温处理1～6 h和24 h时无明显变化。研究表明,DfDREB基因响应激素和非生物胁迫处理,且基因表达受胁迫诱导。该研究结果为进一步探索香鳞毛蕨抗逆分子机制奠定了基础。     

野生鸭儿芹种子休眠特性及破除方法

鸭儿芹种子具有休眠特性,且休眠期长,不经任何处理的种子很难萌发,影响其人工种植。研究了鸭儿芹种子的休眠特性和解除休眠的最佳方法,为我国人工种植野生鸭儿芹提供理论依据。结果表明:TTC法对种子活力的测定表明有活力的种子为(55.33±3.71)%;切破种皮种子与完整种子吸水率在前12 h相差较大,但最终吸水率相差不大,分别达到(70.00±1)%和(68.32±0.32)%,表明种皮并不阻碍种子吸水;种子中存在内源抑制物,其粗提液在较低浓度下即可抑制芹菜种子的萌发;鸭儿芹种子成熟时胚未分化完全,胚率为(28.65±2.488)%,经过低温处理后完成后熟,胚率达到(65.93±3.86)%,萌发率达到100%,因此鸭儿芹种子具有形态生理休眠特性。清水浸种和低温冷藏共同处理可有效解除其休眠,浸种和低温冷藏具有交互效应,浸种36 h、5℃冷藏30d即可解除其休眠,萌发率达到100%,发芽势达到(91.11±0.91)%。已破除休眠的种子适宜其萌发的温度范围扩大(15.0—27.5℃),而且在土壤中也可较好地萌发,萌发率达到(96.67±3.33)%,发芽势达到(71.11±1.93)%。     

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。     

不同低温胁迫下草莓叶片FaGR基因表达分析

为进一步探明草莓谷胱甘肽还原酶(glutathione reductase,GR)基因在低温胁迫下的表达调控模式,以揭示其作用机理和抗逆机制,该研究以草莓栽培品种‘丰香’植株为对象,分别用0、2、4、6、8、10℃低温胁迫以及室温(25℃)作为对照处理24h,从处理植株的新鲜幼嫩叶片中提取总RNA并反转录成cDNA,采用半定量PCR及荧光定量PCR两种定量方法,分析FaGR基因在不同低温胁迫下的表达差异。结果表明:半定量PCR方法与荧光定量PCR方法的结果基本一致。FaGR的相对表达量受到不同低温胁迫和不同程度的诱导,在8℃和10℃低温胁迫下表达量上调,均高于对照水平,且在8℃低温胁迫下相对表达量最高;当温度为6℃时,FaGR表达量急剧下降,当温度低于6℃时,FaGR表达量随着温度的降低而逐渐降低,并低于对照水平;在0℃相对表达量时降至最低水平,约为最大表达量的一半。这说明半定量PCR结果准确可靠,可广泛应用于基因表达研究,而且FaGR表达量受到低温诱导,但不同低温处理对FaGR的表达量诱导程度不同。FaGR相对表达量在一定范围内的低温胁迫下增加,但在此低温范围外的低温胁迫下则降低,该研究表明6℃低温为临界值。以上结果为进一步调控该基因表达提供了科学依据,同时为提高植物抗逆能力提供了基础资料。     

拟南芥AtJ50基因表达特性的研究

以拟南芥野生型和AtJ50∷GUS转基因株系为材料,通过半定量RT-PCR和GUS组织化学染色法探索AtJ50基因的时空表达特性.结果表明:(1)AtJ50基因在根、茎、叶、花和果实中都有表达,花和叶中表达最高,茎和根中次之,成熟的长角果中几乎没有表达;随着植株的成熟和衰老,AtJ50基因的表达量逐渐下降.(2)半定量RT-PCR法分析结果表明,AtJ50基因表达受0.2 mol·L-1的NaCl诱导,随着NaCl处理时间的延长表达量增加,12 h后表达量达到最高,24 h后又有所下降;水分胁迫也可诱导AtJ50基因的表达,水分胁迫0.5 h后AtJ50基因表达量开始增加,1 h后达到最高,2 h后又开始下降.     

凡纳滨对虾ANT2 基因的克隆及低温表达谱分析

为研究凡纳滨对虾适应低温的分子机理, 实验对从低温处理凡纳滨对虾抑制性消减文库中筛选出的一个360 bp EST 序列进行了研究。首先, 同源比对显示该EST 片段与其他物种的ANT2 基因高度同源, 命名为凡纳滨对虾ANT2 基因(LVANT2); 其次, 通过构建凡纳滨对虾肝胰腺全长cDNA 文库, PCR 扩增获得LVANT2 基因的全长cDNA1540 bp, 其中包括1011 bp 的完整开放阅读框, 编码336 个氨基酸残基。然后, 对基因进行了不同组织和低温处理的表达谱分析: (1)组织表达谱的结果显示, 该基因在凡纳滨对虾肌肉组织中表达量最高; (2)在不同低温处理下的表达结果显示, 该基因在15℃处理下基因表达量发生显著变化, 13℃开始呈下调表达, 11℃时表达量又升高; 13℃低温处理不同时间发现该基因在12h 内表达量发生显著变化,48h 后表达量最高。LVANT2 基因的低温诱导表达模式说明其可能在凡纳滨对虾低温适应中发挥作用       

甘菊ClHSP70与ClHSP90基因的克隆及表达分析

该研究以甘菊(Chrysanthemum lavandulifolium)为实验材料,通过RT-PCR方法从甘菊转录组数据中分离出热激蛋白合成相关基因,命名为ClHSP70和ClHSP90。序列分析表明,ClHSP70基因ORF全长为2 559bp,编码852个氨基酸,蛋白功能区预测表明含有典型的HSP70蛋白NBD和SBD保守结构域;ClHSP90基因ORF全长为2 094bp,编码697个氨基酸,含有HATPase结构域和HSP90保守结构域。生物信息学分析表明,甘菊ClHSP70与大豆(Glycine max)和烟草(Nicotiana tomentosiformis)HSP70蛋白有较高的一致性,ClHSP90基因编码的氨基酸序列与紫茎泽兰(Ageratina adenophora)HSP90高度相似;实时荧光定量表达分析表明,在42℃处理不同时间,甘菊叶片中ClHSP70和ClHSP90基因表达均在0.5h时显著增加,1h达到最大值,2h后缓慢下降;不同组织表达分析表明,甘菊在42℃处理1h后,ClHSP70在成熟叶中的表达量显著高于嫩叶和根等其他组织;ClHSP90在成熟茎中的表达量最高。研究说明,ClHSP70和ClHSP90基因具有热激蛋白特征,参与了甘菊热胁迫应答过程,该研究结果为以后深入研究其基因功能奠定了基础。     

刺芹侧耳两种保护酶基因的克隆、分析及温度对其表达量的影响

为了探究温度胁迫下刺芹侧耳中过氧化氢酶和铜锌超氧化物歧化酶基因的作用,本研究通过RT-PCR方法对刺芹侧耳转录组数据库检索得到的过氧化氢酶（CAT）和铜锌超氧化物歧化酶（Cu/Zn-SOD）基因进行克隆,并利用荧光定量PCR分析在高低温胁迫下CAT和Cu/Zn-SOD基因的表达量,以探讨刺芹侧耳中这两种保护酶基因在高、低温胁迫下的响应特征。结果表明,克隆得到的CAT和Cu/Zn-SOD基因开放阅读框长度分别为1 581bp和582bp,分别编码526个和193个氨基酸,分子量分别为59.6kDa、19.62kDa,等电点分别为6.35、6.31。在高温（35℃）和低温（4℃）两组处理中,CAT和Cu/Zn-SOD基因的表达量及酶活均在处理48h时达到最高且与对照组呈现显著差异,说明两种基因在刺芹侧耳抵御高低温胁迫的过程中具有重要作用。本研究结果初步研究了温度胁迫下刺芹侧耳中CAT与Cu/Zn-SOD基因的生物学功能,为进一步解析刺芹侧耳抵抗温度胁迫的机理奠定基础。     

百合LbKCS5和LbKCS17基因的克隆与表达分析

β 酮脂酰辅酶A合成酶(KCS)是超长饱和脂肪酸链(VLCFAs)生物合成中的限速酶,该研究以百合(Lilium brownii var. viridulum Baker.)‘黄天霸’cDNA为模版, 采用RT PCR方法克隆了LbKCS5和LbKCS17基因的CDS序列,并进行序列分析,采用qRT PCR方法分析其基因表达以及胁迫诱导特征。结果表明：(1)LbKCS5和LbKCS17分别包含689和1 238 bp完全阅读框(ORF), 编码186和291个氨基酸;进化分析显示, LbKCS5与油棕KCS5、LbKCS17与莲KCS17序列相似性最高,分别为82.61%和77.62%。(2)基因表达分析结果显示, LbKCS5和LbKCS17 在百合营养器官中均有表达,二者在叶中表达量最高,在根中最少;在花器官中,LbKCS5的表达量在雄蕊中最高,LbKCS17在花药中最高,在花瓣中二者的表达量最低;在花蕾生长发育过程中,花瓣中2个基因的表达均先升高后下降,并于黄蕾期达到峰值;叶中LbKCS5表达量在黄蕾期达到最高,而LbKCS17在叶中的表达整体较低。(3)失水胁迫能提高这2个基因的转录表达,低温诱导LbKCS5表达量的增加,但却降低了LbKCS17的表达。研究表明,LbKCS5和LbKCS17在百合不同器官中均有表达,且均响应失水和低温胁迫,这为研究百合耐失水和低温胁迫以及新品种选育提供了理论支持。     

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.     

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.     

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。     

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.     

Taxonomic notes on Pogonieae ( Orchidaceae ): Cleistesiopsis, a new genus segregated from Cleistes , and description of two new South American species, Cleistes batistana and C. elongata

Summary  A new genus, Cleistesiopsis, was segregated from Cleistes based on morphological and molecular characteristics, and two new species of Cleistes: C. batistana and C. elongata, both occurring in the Brazilian Central Plateau (Central-Western Brazil), are described and illustrated. Furthermore, a key to genera currently recognised within Pogonieae is presented.

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Resumo  Um novo gênero, Cleistesiopsis, é segregado de Cleistes com base em caracteres morfológicos e moleculares, e duas novas espécies de Cleistes: C. batistana e C. elongata, que ocorrem no Planalto Central, no Centro-Oeste do Brasil, s?o descritas e ilustradas. Além disso, uma chave para os gêneros que atualmente comp?em a tribo Pogonieae é apresentada.

     

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.