青稞NBS LRR类基因HvtRGA的克隆与条纹病胁迫表达分析

为探索青稞(Hordeum vulgare L. var. nudum)NBS LRR类基因在青稞抗条纹病中的分子作用机制,该研究以抗条纹病青稞品种‘昆仑14号’和感病品种‘Z1141’为材料,从叶片中克隆了HvtRGA 基因。HvtRGA基因长3 544 bp,包含一个3 306 bp开放阅读框,编码1 101个氨基酸。序列测序后比对发现,‘昆仑14号’与‘Z1141’的碱基序列相似性为99.89%,‘Z1141’的碱基在1196和1945位置处由G替换成A,但氨基酸序列相似性为100%。蛋白质序列分析表明,HvtRGA为亲水性的不稳定酸性蛋白,具有NB ARC保守结构域和5个LRR结构域,属于 NBS LRR 家族。HvtRGA蛋白与大麦的rgaS 9217、rgaS 226编码的NBS LRR氨基酸序列相似性分别为96.55%和88.72%。进化树分析表明,青稞与小麦族的大麦、硬粒小麦和二穗短柄草NBS LRR聚为一个分支,且与大麦 rgaS 9217编码的蛋白亲缘关系最近,其次是大麦rgaS 226编码的蛋白,而与狗尾草和栗的亲缘关系最远。qRT PCR结果表明,条纹病胁迫下,抗病品种和感病品种的HvtRGA基因的表达量极显著升高,且抗病品种‘昆仑14号’感病后基因表达量显著高于感病品种‘Z1141’。研究推测,HvtRGA 基因在青稞抗条纹病的调控过程中可能发挥着重要作用。     

青稞HvnRPS2基因克隆及其在条纹病胁迫下的表达分析

为探索NBS LRR类基因RPS2在青稞抗条纹病过程中的作用,该研究以抗条纹病青稞品种‘昆仑14号’和感病品种‘Z1141’为材料,参照转录组序列设计引物,克隆得到一个差异表达的青稞HvnRPS2基因,进行相应的生物信息学分析,并采用实时荧光定量PCR(qRT PCR)法分析HvnRPS2基因在条纹病侵染下不同抗性青稞品种的表达模式。结果表明：(1)HvnRPS2基因全长3 089 bp,无内含子,包含1个2 760 bp的开放阅读框,编码919个氨基酸,理论等电点为5.93,预测蛋白分子量为104.2 kD,其编码的蛋白为亲水性蛋白,二级结构主要由无规则卷曲和ɑ 螺旋组成。(2)蛋白质多序列比对及进化树分析表明,HvnRPS2含有高度保守的NB ARC和LRR结构域,属于NB ARC蛋白家族成员,与大麦HvRPS2和水稻OsRPS2亲缘关系最近。(3)qRT PCR分析显示,随着条纹病感病时间的延长,青稞HvnRPS2基因表达量呈先降低后升高再降低的模式;与正常叶片相比,感病叶片的HvnRPS2基因表达量显著下调,且感病品种表达量下调值显著低于抗病品种(P＜0.01)。研究认为,HvnRPS2在青稞抗条纹病过程中发挥重要的负调控作用。研究结果为进一步探究该基因在青稞抗条纹病中的调控机理奠定基础。     

葡萄抗白粉病相关基因γVPE的克隆与表达分析

该研究采用RT-PCR技术,从抗病的中国野生华东葡萄‘白河-35-1’和感病的欧洲葡萄‘佳丽酿’中克隆了液泡加工酶基因(γVPE),分别命名为VpγVPE和VvCγVPE。克隆的2个γVPE基因cDNA长度均为1 624bp,ORF为1 482bp,编码493个氨基酸。氨基酸多序列对比分析发现,‘白河-35-1’、‘佳丽酿’、‘无核白’和‘黑比诺’葡萄中的γVPE基因底物结合口袋域的3个关键氨基酸之一的丝氨酸(Ser395)均变为丙氨酸(Ala),与其他植物的VPE基因底物口袋结合域有所不同。实时荧光定量PCR表明,在白粉菌诱导后的不同时期内,γVPE基因在感病葡萄和抗病葡萄中的表达模式不同,抗病株系中VpγVPE基因的表达量在诱导后的前期(4h和48h)和后期(168h)均有所增加,而感病株系中VvCγVPE基因在诱导后4h表达量最高,随后降低。γVPE基因在白粉菌诱导后不同时期内表达量的变化,表明γVPE基因在一定程度上与葡萄的抗性相关。研究结果为进一步揭示γVPE基因在抗病过程中的分子机理奠定了基础。     

石榴木质素合成相关基因PgMYB308的克隆和表达分析

该研究以‘红玉石籽’(Punica granatumcv.Hongyushizi)石榴为试验材料,采用RACE和RT-PCR方法,获得与木质素的合成相关基因PgMYB308。PgMYB308基因cDNA全长792bp,编码263个氨基酸。分子量为29.56kD,理论等电点为9.07。序列比对和功能域分析发现,PgMYB308包含R2和R3保守域,以及C1、C2、C4和锌指基序。系统进化树分析显示,PgMYB308与其他物种起源相同,而与桉树EgMYB308亲缘关系最近。荧光定量PCR分析表明,PgMYB308在茎、叶和种子等组织中均有表达,其中茎中表达量最高,叶片中表达量最低;PgMYB308在‘突尼斯软籽’中表达最高,而在‘红玉石籽’和‘白玉石籽’中表达量较低;PgMYB308在‘红玉石籽’籽粒的不同时期均有表达,但在花后20d相对表达量最高,以后随发育进程呈现逐渐下降的趋势。     

百合LbCAT和LbGPX基因的克隆与表达分析

以切花百合(Lilium brownii var. viridulum)‘卡瓦纳’cDNA为模板,克隆了过氧化氢酶(LbCAT)和谷胱甘肽过氧化物酶(LbGPX)基因。序列分析表明,这2个基因分别包含1 479 bp和519 bp的开放阅读框(ORF),编码492个和172个氨基酸。进化分析结果表明,LbCAT蛋白与岷江百合CAT蛋白的氨基酸序列相似性最高(99.19%),且亲缘关系最近;LbGPX蛋白与油棕GPX蛋白的氨基酸序列相似性最高(78.61%),亲缘关系最近。qRT PCR结果显示,LbCAT和LbGPX在百合根、鳞茎、叶和花中都有表达。LbCAT在叶中表达量最高,LbGPX在花中表达量最高。这2个基因在百合花蕾的生长发育过程中均有表达,且表达量逐渐增加;在PEG处理后2个基因的转录水平升高,但独角金内酯(SLs)处理却显著降低了这2个基因的转录水平;该结果为百合抗逆性机理研究以及抗逆育种奠定了基础。     

‘黄花’梨及其芽变‘绿黄花’梨HHT基因克隆与表达分析

该试验以砂梨品种‘黄花’梨（果皮褐色）及其芽变‘绿黄花’梨（果皮绿色）盛花后第8周的果皮为试材,利用常规PCR和巢式PCR技术克隆了ω 羟基棕榈酸O 阿魏酰转移酶（ω hydroxypalmitate O feruloyl transferase, HHT）基因cDNA的全长,命名为 PpyHHT（登录号为KX131155）。序列分析结果表明,该基因开放阅读框（ORF）为1 335 bp,编码444个氨基酸。生物信息学分析显示,推定的PpyHHT蛋白质相对分子质量为49.91 kD,等电点是4.75,与白梨相似性高达98%,亲缘关系最近。实时荧光定量PCR（qRT PCR）表达分析显示,2种梨果皮中 PpyHHT基因在盛花后6~9周的4个转色关键期表达量不断变化,在‘黄花’梨果皮中的表达量明显高于‘绿黄花’梨。推测 PpyHHT基因可能参与砂梨果实褐色/绿色性状的形成。     

石榴4-香豆酸辅酶A连接酶基因的克隆和表达分析

4-香豆酸辅酶A连接酶(4-coumarate-CoA ligase,4CL)是木质素合成途径的关键酶。该研究以‘红玉石籽’(Punica granatum cv.Hongyushizi)石榴为试验材料,采用RACE和RT-PCR方法获得4CL同源基因Pg4CL。Pg4CL基因cDNA全长2 121bp,编码544个氨基酸;序列比对和功能域分析发现,Pg4CL包含有2个保守域SSGTTGLPKGV和GEICIRG;系统进化树分析显示,Pg4CL与其他物种起源相同,而与赤桉Ec4CL亲缘关系最近。荧光定量PCR分析表明,Pg4CL基因在叶、果皮、种皮和茎等组织中均有表达,其中果皮和叶片中表达量较高,种皮中表达量最低;Pg4CL基因在6个石榴品种的种皮中均有表达,其中在‘突尼斯软籽’中表达较高,‘会理软籽’表达量最低;Pg4CL基因在‘红玉石籽’籽粒的不同时期均有表达,在15~60d相对表达量逐步上升,并在60d时达到最高;75d以后表达量急剧下降,仅维持较低水平表达。     

水芹肉桂醇脱氢酶基因的克隆与表达特性分析

以‘八卦洲水芹’及其紫色叶柄突变型水芹‘南选八卦洲紫水芹’为实验材料,利用RT-PCR方法从‘南选八卦洲紫水芹’中克隆得到水芹肉桂醇脱氢酶(CAD)基因,命名为OjCAD。OjCAD基因开放阅读框长为1 074bp,编码357个氨基酸。OjCAD蛋白相对分子质量为39 143.10,理论等电点为6.91,属于MDR家族。系统进化分析显示,水芹OjCAD与同属伞形科的胡萝卜CAD进化关系最近,具有高度保守性。OjCAD编码的蛋白属于疏水蛋白,空间结构主要由7个α-螺旋和17个β-折叠组成。实时定量PCR分析显示,OjCAD基因在紫色和非紫色水芹的叶片和叶柄中相对表达量存在差异,水芹OjCAD基因在叶片中的表达量显著高于叶柄,在‘八卦洲水芹’中的表达量高于‘南选八卦洲紫水芹’。该研究结果为进一步分析水芹木质素生物合成奠定了基础。     

刺葡萄抗白腐病转录因子VdWRKY53基因克隆及表达

利用同源克隆的方法,分别获得‘刺葡萄0943’的转录因子VdWRKY53基因的编码区和启动子区域,分析其序列特征,通过实时荧光定量检测其对白腐病菌侵染和水杨酸诱导的反应,并以感病品种欧亚种‘黑比诺’为对照,分析不同种质中转录因子VdWRKY53基因序列及表达差异。结果表明:‘刺葡萄0943’的VdWRKY53基因,在其编码区和启动子区域都有抗病基因的序列和位点特征,在DNA和氨基酸水平上与‘黑比诺’VvWRKY53有5处差异,这5处氨基酸差异可能造成了其功能的差异;‘刺葡萄0943’和‘黑比诺’中的WRKY53基因启动子受葡萄白腐病菌和水杨酸诱导后表达量增加,且VdWRKY53基因表达量和趋势不同于‘黑比诺’VvWRKY53。生物信息学分析和实时荧光定量表明,在‘刺葡萄0943’抗病途径中VdWRKY53转录因子具有重要的生物学作用。     

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。     

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.     

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.     

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 the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

马齿苋内生菌橘青霉和波兰青霉中抗青枯菌的活性物质

为了挖掘农作物病害生物防治新资源,以药用植物马齿苋(Portulaca oleracea)为材料,通过培养基种植法分离和纯化其根、茎、叶中的内生菌,以青枯菌(Ralstonia solanacearum)的抑菌试验评价其活性,采用菌落形态观察和ITS序列分析鉴定菌种。结果表明,从马齿苋筛选出2种具有抑制青枯菌的内生菌橘青霉(Penicillium citrinum)和波兰青霉(P. polonicum),采用液相与四极杆飞行时间串联质谱(UPLC-QTOF-MS)鉴定2种内生菌的主要活性物质为橘霉素,其对青枯菌的抑制效果比链霉素更好。因此,这为植物青枯病的生物防治提供科学依据。