苹果酸度相关基因MdPH1的克隆、表达及亚细胞定位分析

以苹果属(Malus)植物沧江海棠(M. ombrophila Hand.-Mazz)的果实为材料,对其发育过程中苹果酸的含量进行测定,并结合转录组测序的方法筛选控制果实酸度的候选基因。结果显示:MdPH1候选基因的编码区包含2829 bp,编码942个氨基酸;基因组序列全长为4269 bp,包含8个外显子和7个内含子。对10份苹果种质资源中PH1基因序列的分析结果表明,该基因序列中存在22个单核苷酸多态性(SNP),其中13个位于内含子区,9个位于外显子区;位于最后一个外显子上SNP(G/A)的变异导致了编码氨基酸从缬氨酸变为异亮氨酸。MdPH1蛋白包含8个跨膜结构域,其中蛋白N端包含3个跨膜结构域,C端包含5个跨膜结构域。系统进化分析结果显示,苹果中的PH家族成员与梨(Pyrus communis L.)中的PH家族成员聚集成一簇。组织特异性表达结果发现,MdPH1基因在苹果果实中的表达量最高,其次是叶、花和根,茎中表达量最低。亚细胞定位分析表明MdPH1蛋白定位于液泡膜上。     

苹果酸度相关基因MdPH1的克隆、表达及亚细胞定位分析

以苹果属（Malus）植物沧江海棠（M.ombrophila Hand.-Mazz）的果实为材料,对其发育过程中苹果酸的含量进行测定,并结合转录组测序的方法筛选控制果实酸度的候选基因。结果显示：MdPH1候选基因的编码区包含2829 bp,编码942个氨基酸;基因组序列全长为4269 bp,包含8个外显子和7个内含子。对10份苹果种质资源中PH1基因序列的分析结果表明,该基因序列中存在22个单核苷酸多态性（SNP）,其中13个位于内含子区,9个位于外显子区;位于最后一个外显子上SNP（G/A）的变异导致了编码氨基酸从缬氨酸变为异亮氨酸。MdPH1蛋白包含8个跨膜结构域,其中蛋白N端包含3个跨膜结构域,C端包含5个跨膜结构域。系统进化分析结果显示,苹果中的PH家族成员与梨（Pyrus communis L.）中的PH家族成员聚集成一簇。组织特异性表达结果发现,MdPH1基因在苹果果实中的表达量最高,其次是叶、花和根,茎中表达量最低。亚细胞定位分析表明MdPH1蛋白定位于液泡膜上。     

杨柳田头菇B交配型基因信息素受体片段的克隆与分析

利用简并PCR及DNA步移法,从杨柳田头菇Agrocybe salicacola YAASM0711菌株中扩增得到了一个4 231 bp的核酸片段.经过比对及序列预测,所获得序列中含有杨柳田头菇交配型编码基因中的信息素受体部分,其序列长度为1194 bp,包含4个内含子,5个外显子的长度分别为217 bp,113 bp,67 bp,138bp,449 bp.拼接后的ORF全长984 bp,编码327个氨基酸残基.该序列与灰盖鬼伞Coprinus cinerea、双色蜡蘑Laccaria bicolor信息素受体氨基酸序列较为相似,含有7个跨膜区.信息素受体遗传进化分析显示,其与多个物种信息素受体聚集在一起,可能与真菌信息素受体的多种起源有关.     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

百合查尔酮合成酶基因的克隆与分析

以西伯利亚百合为试材,通过半巢式PCR和RT-PCR技术分别克隆了查尔酮合成酶基因(CHS)的DNA和cDNA.生物信息学分析显示,CHS的DNA序列全长1 397 bp(登录号HM622754),包含2个外显子和1个内含子;cDNA序列编码区全长1 182 bp(登录号HQ161731),编码393个氨基酸,具有3个典型的CHS蛋白结构域:N-末端结构域(Lys3-Pro229)、C-末端结构域(Gln239-Pro389)和聚合酶Ⅲ结构域(Met1-Thr391);不同百合品种的CHS基因编码的氨基酸序列相似性高达98%,表明百合CHS基因在进化上呈现出十分保守的趋势;不同植物CHS基因序列的系统进化邻接树结果表明:百合与单子叶植物鸢尾及禾本科的水稻、大麦、玉米等亲缘关系更为接近.     

不结球白菜BcHSP70-1基因的克隆与进化及其表达分析

利用已报导的拟南芥和甘蓝型油菜同源基因序列设计克隆引物,从不结球白菜中克隆了BcHSP70-1基因。在BcHSP70-1基因测序后,对该基因编码蛋白质的理化性质、亚细胞定位和跨膜结构域等生物信息学分析结果表明,BcHSP70-1编码的蛋白质是亲水蛋白,定位在细胞质中,该基因ORF全长1 950bp,编码649个氨基酸,有1个内含子和2个外显子,内含子为324bp;对BcHSP70-1直系同源基因外显子和内含子比对分析显示,内含子的差异更显著。实时定量PCR表达分析表明,BcHSP70-1的表达量在不同耐热性的栽培种中有较大差异,在不耐热的不结球白菜品种(NHCC002)中未见该基因组成性表达;与38℃高温胁迫相比,4℃低温胁迫下诱导的BcHSP70-1表达量更高。     

丹参ACC氧化酶基因(SmACO1)的克隆与序列分析

依据丹参转录组数据库序列信息,采用RT-PCR和染色体步移技术从丹参中首次克隆得到ACC氧化酶基因,命名为SmACO1(GenBank注册号为JQ026111)。该基因gDNA序列长1 347 bp,由3个外显子和2个内含子组成;cDNA全长1 117 bp,包含945 bp的开放阅读框,编码314个氨基酸残基。生物信息学分析显示SmACO1为无信号肽与跨膜结构域,且定位于细胞质的稳定亲水蛋白,含有Fe²⁺依赖的加氧酶结构域。实时荧光定量PCR结果表明,SmACO1基因在丹参不同组织器官中差异表达,花中表达量最高;其表达受到病原菌和茉莉酸甲酯的诱导,表明SmACO1基因可能在植物防御反应中发挥作用。     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

草鱼腺苷酸基琥珀酸裂解酶克隆及序列分析

腺苷酸基琥珀酸裂解酶(Adenylosuccinate lyase,ADSL)是嘌呤核苷酸合成过程中的关键酶.研究以草鱼(Ctenopharyngodon idellus)肠道cDNA文库为基础,应用PCR、RT-PCR和RACE技术,成功获得了草鱼肠道组织腺苷酸基琥珀酸裂解酶基因的cDNA全长和基凶组DNA全长.该基因全长1584 bp,包含一个1449 bp的开放阅读框,编码482个氨基酸,与其他脊椎动物比对显示,其序列具有较高的保守性.草鱼腺苷酸基琥珀酸裂解酶基因组DNA由13个外显子和12个内含子组成,其外显子拼接位点非常保守.遵循GT-AG原则.     

尾叶桉GLU4无性系F5H基因的克隆表达及序列分析

[目的]克隆尾叶桉(Eucalyptus urophylla)GLU4中F5H基因全长,并对其进行序列和表达模式分析。[方法]根据NCBI公布的F5H基因保守序列设计引物,以尾叶桉GLU4茎部组织为试验材料克隆其g DNA和c DNA片段,利用荧光定量PCR分析其在植株不同组织中的表达模式。[结果]该基因g DNA长2 358 bp,c DNA长1 610 bp,含有2个外显子、1个内含子。开放阅读框编码529个氨基酸,Eu F5H编码的蛋白为一个带负电荷、存在跨膜结构、定位在内质网起始分泌途径中的蛋白质,二级结构包含典型的的α-螺旋和β-折叠,根据三级结构推测其可能具有羟化酶活性。Eu F5H蛋白与蓝桉亲缘关系较近,Eu F5H在不同组织中表达水平差异显著,其中半木质化茎中表达水平相对最高。[结论]成功获得了尾叶桉GLU4木质素合成酶基因F5H的全长序列。     

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.     

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.     

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.