干旱胁迫下棉花幼苗转录因子BES1/BZR1对外源油菜素内酯的响应表达特征

植物激素油菜素内酯(Brassinosteroid,BR)具有提高植物抗旱性的作用,该研究探讨干旱胁迫下外源喷施BR对棉花干旱胁迫响应基因表达的影响。采用PCR方法从棉花‘新陆早17号’幼苗克隆获得1个干旱胁迫响应转录因子基因,命名为GhBES1/BZR1(GenBank登录号KP272000)。序列分析表明GhBES1/BZR1基因开放阅读框为960bp,编码319个氨基酸,理论分子量为34.3kD,理论等电点为8.95。保守结构域分析显示,该基因编码的蛋白具有一个DUF822保守结构域。利用2.5%PEG-6000对棉花‘新陆早17号’幼苗进行干旱胁迫处理24h,再分别喷施水、BR和Z(BR抑制剂),qRT-PCR分析结果表明,PEG-6000干旱胁迫下用BR处理3h后,GhBES1/BZR1基因表达量明显提高,当BR处理6和12h时,GhBES1/BZR1基因的表达量较3h时明显下降。研究认为,在干旱胁迫下对棉花喷洒BR,GhBES1/BZR1基因能够快速表达以响应干旱胁迫,外源喷施BR有助于提高棉花的抗旱能力。     

XTH基因家族在‘妃子笑'荔枝花穗不同处理方式的表达分析

木葡聚糖内切葡聚糖酶/水解酶(xyloglucan endotransglucosylase/hydrolase,XTH)是一类重要的糖苷水解酶,在促进植物细胞壁延展以及响应逆境胁迫方面发挥着重要作用。为研究荔枝XTH家族基因在不同花穗处理方式下的表达情况,该文以‘妃子笑’荔枝为实验材料,对‘妃子笑’花穗进行疏花和喷施烯效唑处理;基于前期转录组数据库鉴定出29个LcXTH家族成员,利用荧光定量PCR(RT-qPCR)技术对LcXTH基因家族成员在不同处理后花穗的表达进行研究,分析XTH家族基因对不同花穗处理方式的响应情况。结果表明:LcXTH家族基因成员表达在对照、疏花处理和烯效唑处理后花穗不同发育阶段表达规律不同,且不同基因存在表达量的差异。其中LcXTH亚家族Ⅲ、LcXTH亚家族Ⅳ(LcXTH2、LcXTH3、LcXTH4、LcXTH5、LcXTH6、LcXTH7、LcXTH9、LcXTH10、LcXTH11、LcXTH12、LcXTH13、LcXTH22和LcXTH23)成员及LcXTH20表达量较高,推测在花穗发育中起关键作用。‘妃子笑’花穗疏花处理14 d后LcXTH家族大部分成员表达上调,推测疏花对LcXTH的表达起到正调控作用;‘妃子笑’花穗喷施烯效唑后LcXTH家族成员的表达下调,推测烯效唑处理对LcXTH的表达起到负调控作用。     

海岛棉GbGSTU7的克隆及其对枯萎病菌侵染的应答

该研究基于前期转录组测序数据分析,采用RT-PCR方法从海岛棉06-146中克隆得到参与海岛棉抗枯萎病调控的候选基因谷胱甘肽转移酶基因(GbGSTU7),对其序列进行生物信息学分析,并对GbGSTU7基因在病菌侵染下不同组织的表达特性进行分析,以探讨GbGSTU7基因与棉花抗枯萎病的关系。结果显示:(1)GbGSTU7基因CDS全长711 bp,编码236个氨基酸,属于GST家族tau类,二级结构由5个β折叠和9个α螺旋构成,三级结构呈球状。(2)枯萎病菌侵染后,GbGSTU7基因表达量在抗枯萎病品种06-146的根中呈下降趋势,48 h达到最低,茎和叶中呈先升高再降低的趋势,在茎中12 h达到最高,在叶中24 h达到最高;在感枯萎病品种‘新海14’的根中呈先下降再上升趋势,且在8 h达到最低,在茎和叶中的趋势无明显规律,但在茎中8 h达到最高,在叶中4 h达到最高;且两个材料中均表现为GbGSTU7基因在根中的表达量最低时,茎的表达量升到最高。研究表明,GbGSTU7基因在海岛棉抗、感病材料的各组织中均可响应枯萎病菌的诱导表达,且均具有明显的组织特异性;推测GbGSTU7基因可能与棉花抗枯萎病有着密切的关系,可能在棉花抵御病菌侵染的过程中发挥重要的调控作用。     

龙眼体胚发生过程中DlAGO4基因的克隆及表达分析

该研究基于龙眼基因组数据库,采用RT-PCR技术,以‘红核子’品种龙眼松散型胚性愈伤组织cDNA为模板,进行龙眼胚性愈伤组织DlAGO4基因的克隆和生物信息学分析,并采用实时荧光定量技术分析其在龙眼体细胞胚胎发生不同阶段、不同组织部位、激素和非生物胁迫处理以及5-氮胞苷(5-azac)处理的表达模式。结果表明:(1)DlAGO4基因cDNA全长为3 425 bp,包含开放阅读框长度为2 781 bp,编码926个氨基酸。(2)生物信息学分析表明,DlAGO4蛋白为碱性亲水非分泌蛋白,含有AGO经典的保守结构域PAZ和Piwi,与克莱门柚CcAGO4的同源性最近,含有85个磷酸化位点和2个糖基化位点;亚细胞定位预测其最可能定位于细胞核;MicroRNA预测显示,DlAGO4基因受到4个miRNA靶向调控。(3)qRT-PCR结果表明,DlAGO4在龙眼球形胚阶段和种子中相对表达量最高;激动素、水杨酸、NaCl、甘露醇、PEG-4000和ABA处理均能促进DlAGO4基因的表达,而2,4-D和MeJA处理抑制其表达;不同浓度的5-azac处理1 d和3 d抑制DlAGO4的表达,但从处理第6天开始该基因呈上调表达,并于处理第12天相对表达量最高。研究认为,DlAGO4基因可能参与龙眼球形胚和种子的转录调控,且可能参与KT、SA的激素信号转导途径和NaCl、甘露醇、PEG-4000、ABA的逆境胁迫响应途径以及DNA甲基化调控机制。     

茶树TIFY基因家族鉴定及非生物胁迫下表达分析

TIFY基因家族在茶树激素信号转导及其逆境胁迫具有十分重要的作用。该文利用生物信息学方法对茶树基因组中的TIFY家族进行了鉴定,分析其理化性质、系统进化、基因结构、染色体定位、启动子区顺式作用元件、组织表达模式,并通过RT-qPCR对部分TIFY家族成员进行了非生物胁迫。结果表明:(1)茶树TIFY基因家族成员19个(CsTIFY1～CsTIFY19),分属于4个蛋白亚家族TIFY、JAZ、ZML 和 PPD,且不均匀地分布在8 条染色体上,按照进化关系及结构特点可分为 7 组,每组内具有相似的基因结构与保守基序组成。(2)CsTIFYs基因的启动子区具有多种包含激素和非生物胁迫响应的顺式作用元件,通过实时荧光定量(RT-qPCR)分析其家族成员对在茉莉酸甲酯、盐(20%氯化钠)、冷(4 ℃)以及干旱(20% PEG-6000)处理下反应强烈,部分基因在根与顶芽中有较高的表达量。由此推测,TIFY基因家族可能在茶树激素信号调控、胁迫响应、生长和发育等多方面发挥功能作用。     

茶树CsCIGR基因克隆及表达特性分析

该研究以茶树基因组数据库为基础,采用RT-PCR技术,从茶树‘龙井43’中克隆得到基因CsCIGR。序列分析显示,CsCIGR基因开放阅读框长度为1 677 bp,编码588个氨基酸。进化分析表明,CsCIGR属于GRAS家族的PAT1亚家族。多序列比对显示,茶树CsCIGR蛋白与其他植物的GRAS蛋白氨基酸序列具有很高的相似性。氨基酸理化性质分析显示,CsCIGR转录因子属于亲水性蛋白。亚细胞定位预测显示,CsCIGR可能位于细胞核中。启动子预测分析发现,CsCIGR启动子区域包含胁迫响应元件(STRE)、干旱应答元件(MYC)、厌氧诱导元件(ARE)等多种与逆境响应相关的顺式作用元件。荧光定量PCR分析结果显示,CsCIGR基因在低温(4℃)、高温(38℃)、干旱(200 g·L~(-1) PEG)、高盐(200 mmol·L~(-1) NaCl)胁迫下均能诱导表达,且对高盐,低温和高温胁迫响应更为明显,推测CsCIGR基因在茶树响应逆境胁迫中发挥重要作用。该研究为茶树抗性育种筛选基因提供了重要理论依据。     

茶树BES1转录因子全基因组鉴定与分析

植物特异性转录因子(BRI1-EMS-SUPPRESSOR1,BES1)家族参与调节油菜素内酯(brassinosteroids,BR)信号通路,在植物生长和应对非生物胁迫的反应中发挥重要作用。该研究利用生物信息学方法对茶树BES1转录因子家族进行鉴定,分析茶树CsBES1基因在8个茶树组织中的表达模式,并对CsBES1转录因子家族在低温、干旱及ABA激素处理下的表达进行分析,以揭示茶树CsBES1转录因子家族的功能及其对环境胁迫的响应。结果显示:(1)从茶树全基因组中鉴定获得10个茶树BES1转录因子家族成员,均具有完整的BES1_N结构域;根据系统进化关系将10个CsBES1转录因子家族分成3组,分别包含2个、3个和5个成员;该家族成员每个基因含有2～11个外显子。(2)组织表达模式分析显示,茶树BES1转录因子家族在生长活跃的茶树嫩梢和成熟叶中表达量较高,不同成员之间表达存在差异性。(3)上游启动子区域分析发现大量与植物激素和非生物胁迫响应相关的顺式作用元件。(4)荧光定量检测发现,BES1基因在茶树不同胁迫处理下的表达模式不同,在低温和脱落酸处理下,CsBES1-1、CsBES1-7、CsBES1-8和CsBES1-9基因的表达量显著提高,而在干旱处理下,CsBES1-2、CsBES1-4、CsBES1-5、CsBES1-6、CsBES1-7、CsBES1-8和CsBES1-9基因的表达量显著提高。     

棉花NHL基因家族鉴定及黄萎病菌胁迫下的表达分析

已有研究证明拟南芥NHL(NDR1/HIN1-like)基因在抗病中发挥作用。本研究从海岛棉和陆地棉中分别鉴定出100个和118个NHL基因,并对其进行了生物信息学分析。海岛棉和陆地棉NHL基因家族成员几乎遍布各条染色体,并可分为3个亚组,都含有3个与拟南芥保守基序相似的保守结构域。利用接菌后抗病陆地棉品种的转录组数据,分析NHL基因家族各成员表达变化,发现在黄萎病菌胁迫下,有57个陆地棉NHL基因在根和茎中的表达都发生了明显的变化,其表达模式可分为3种类型:接菌前高,接菌后表达下降;接菌前较高,接菌后表达先降后升;接菌前低,接菌后表达升高。第一类基因的表达在接菌后一直受到抑制,推测其在棉花抗黄萎病反应中受到负调控;后两类基因受黄萎病菌诱导表达升高,推测其在棉花抗病中受到正调控。本研究结果为分析成员较多的NHL基因家族在棉花抗黄萎病中的作用提供了理论依据。     

‘潘那利’番茄碱性螺旋环螺旋转录因子基因的克隆与表达分析

碱性螺旋环螺旋(basic/Helix Loop Helix, bHLH)转录因子是植物最大的转录因子家族之一,其广泛参与植物逆境胁迫响应。该研究从野生‘潘那利’番茄(Solanum pennellii Correll)中成功克隆出bHLH转录因子基因SpbHLH89(Sol Genomics登录号Sopen04g001150),采用qRT PCR分析其在干旱胁迫下的表达模式,并利用异源表达初步分析其对非生物胁迫的响应。结果表明：(1)SpbHLH89编码区包含684 bp,编码227个氨基酸,具有典型的碱性螺旋环螺旋区,主要定位于细胞核中;进化树结果显示,SpbHLH89转录因子高度保守,与拟绒毛烟草NtbHLH94(Nicotiana tomentosiformis)存在高度相似性。(2)qRT PCR结果显示,SpbHLH89在‘潘那利’番茄的茎、叶和花中均有表达,其表达量受干旱胁迫诱导。(3)SDS PAGE与Western bloting结果显示,pET 30a SpbHLH89重组蛋白大小约为31 kD。(4)在盐胁迫(400 mmol/L NaCl)和干旱胁迫(600 mmol/L甘露醇)条件下,异源表达重组蛋白的E. coli BL21(DE3)重组菌生长速度提高,说明异源表达SpbHLH89转录因子基因可提高细菌对非生物胁迫的耐受性。     

海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证

在对黄萎病菌胁迫处理的海岛棉Pima 90-53根组织全长c DNA文库分析中,筛选到一个与黄萎病胁迫相关的杂合富含脯氨酸蛋白(hybrid proline-rich protein)基因,将其命名为Gb Hy PRP1。该基因c DNA序列全长1747 bp,开放阅读框945 bp,编码一个由314个氨基酸残基组成的蛋白,包含信号肽、N端富含脯氨酸域及C端Pollen Ole e I域。同源序列分析显示,Gb Hy PRP1与来自雷蒙德氏棉、陆地棉和亚洲棉的Hy PRP1蛋白序列相似性最高,分别为95.95%、93.87%和91.34%。q RT-PCR分析结果显示,受黄萎病菌胁迫后海岛棉根部Gb Hy PRP1表达显著下调。将Gb Hy PRP1基因克隆至植物超表达载体,农杆菌介导转化拟南芥获得转基因植株。病指统计分析表明Gb Hy PRP1过量表达显著降低了拟南芥对黄萎病的抗性。据此推测Gb Hy PRP1参与棉花抗黄萎病,可能是一个重要的负调控因子。     

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.     

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.     

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.     

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.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

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.