外源亚精胺对淹涝胁迫下黄瓜根HSP70表达的影响

采用逆转录聚合酶链式反应(RT-PCR)及蛋白免疫印迹杂交(Western Blot)技术,研究0.5 mmol/L亚精胺浸种的黄瓜幼苗在淹水胁迫下,根热激蛋白70基因(HSP70)mRNA和蛋白质的表达量的变化。结果表明:淹水胁迫使黄瓜根HSP70的mRNA和蛋白的表达呈现先上升后下降的趋势,在淹水4 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理; 亚精胺浸种的黄瓜根HSP70的mRNA和蛋白的表达量在24 h内呈一直上升的趋势,在淹水24 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理。淹涝胁迫下,亚精胺浸种的黄瓜根HSP70的mRNA和蛋白表达量在淹水12 h和24 h时极显著高于蒸馏水浸种。外源亚精胺能诱导淹涝胁迫下黄瓜幼苗根HSP70 mRNA和蛋白质的表达量的增加,缓解淹涝胁迫对黄瓜造成的伤害。     

忽地笑LaSUVH1基因克隆与功能分析

为了深入探究忽地笑(Lycoris aurea)组蛋白赖氨酸甲基转移酶(histone lysine methyltransferase, HKMT)基因的功能,该研究根据前期转录组测序结果,采用RT PCR方法克隆得到一个组蛋白赖氨酸甲基转移酶基因LaSUVH1。序列分结果表明, LaSUVH1基因的编码区(coding sequences, CDs)序列长2 007 bp,编码668个氨基酸残基;LaSUVH1蛋白不具有信号肽结构,无跨膜结构,为亲水性蛋白,含有SET、YDG/SRA、Pre SET和Post SET结构域;序列比对和系统进化树分析发现,LaSUVH1与芦笋AoSUVH1 like蛋白亲缘关系最近。实时荧光定量PCR分析表明,LaSUVH1基因在忽地笑不同组织部位均有表达,且在叶中表达量最高。经潮霉素筛选成功获得3个反义过表达LaSUVH1的转基因拟南芥株系。进一步功能分析发现,反义过表达LaSUVH1促进了拟南芥幼苗侧根的发生,降低了拟南芥对NaCl的耐受性,增加了拟南芥种子萌发对脱落酸(ABA)的敏感性,表明LaSUVH1基因响应盐胁迫应答可能依赖ABA信号通路。     

中国南瓜CmNPR1基因的克隆和表达分析

从实验室前期对中国南瓜雌花败育转录组测序结果推测,CmNPR1基因可能在南瓜花发育过程中发挥重要功能。该研究以中国南瓜自交系‘3 1’为试验材料,采用同源克隆方法获得中国南瓜CmNPR1基因CDS序列,通过生物信息学、基因表达以及亚细胞定位分析对该基因进行初步研究, 为进一步研究CmNPR1基因在南瓜花发育中的功能和作用机制奠定基础。结果表明：(1)中国南瓜CmNPR1基因CDS全长1 442 bp,编码480个氨基酸;蛋白序列包含有一个BTB/POZ和一个锚蛋白重复序列(Ank)保守结构域;该蛋白无信号肽和跨膜结构;多序列比对分析结果显示,CmNPR1氨基酸序列与美洲南瓜的亲缘关系最近,为96.05%,其次是印度南瓜,为95.63%。(2)CmNPR1基因在所取样品中花纵径0.5 cm时期表达量最高,且在花不同结构中柱头的表达量最高。(3)通过拟南芥原生质体亚细胞定位分析发现,该蛋白定位于细胞质和细胞核。     

小麦TaDRLea3 2基因的克隆及胁迫诱导表达分析

胚胎发育后期丰富蛋白(late embryogenesis abundant protein,LEA蛋白) 是植物体中广泛存在的一类与渗透调节相关的家族蛋白,植物受非生物胁迫会大量表达。该研究采用同源克隆技术,从干旱诱导的小麦品种‘郑引1号’ (Triticum aestivum L.)中获得1个新的LEA3家族基因（ TaDRLea3 2）,该基因全长668 bp,编码区为570 bp,编码189个氨基酸。生物信息学分析表明该蛋白为亲水性蛋白,二级结构以α 螺旋为主,含有9个由11个氨基酸组成的保守结构域,为典型的LEA3蛋白,存在3个磷酸化位点,无信号肽结构域及跨膜结构域,可能定位于细胞质中;实时定量PCR结果表明, TaDRLea3 2基因受干旱、高盐、低温诱导表达,同时也受外源ABA诱导,推测 TaDRLea3 2为ABA依赖型 LEA3基因,以不同机制参与小麦对非生物胁迫的应答过程,为深入分析小麦LEA3家族蛋白的抗逆机制奠定了基础。     

草菇热激蛋白60基因(Vvhsp60)生物信息学分析及低温下的表达

【背景】生物受到温度胁迫时,热激蛋白被诱导并在短时间内大量产生,可以使受损的蛋白质恢复正常构象,增强生物对逆境胁迫的耐受性。【目的】初步探究草菇热激蛋白60(Vvhsp60)与低温耐受性的关系,为深入开展草菇不耐低温特性的遗传改良奠定理论基础。【方法】对Vvhsp60进行生物信息学分析,以低温敏感型草菇菌株V23及耐低温菌株VH3为实验材料,利用实时荧光定量PCR技术分析低温胁迫及热激诱导后在低温下草菇菌丝体中Vvhsp60基因的表达水平。【结果】草菇Vvhsp60编码蛋白不存在信号肽,不属于分泌蛋白,在线粒体和细胞质内发挥生物学作用,属于双向跨膜蛋白。低温处理显著提高了V23与VH3菌丝体中Vvhsp60基因的表达量,而且VH3中的表达量显著高于V23,推测Vvhsp60基因的表达量高可能有助于增强草菇对低温胁迫的耐受性。经热激处理后两菌株Vvhsp60基因的表达量显著高于各自未热激处理的对照组,表明热激处理可诱导Vvhsp60基因的表达。【结论】Vvhsp60与草菇低温耐受性相关,并且热激可以诱导Vvhsp60基因的表达。     

蔗茅EfMYB1基因的克隆与表达分析

为了充分利用蔗茅(Erianthus fulvus)野生资源,挖掘其优良的抗性基因,丰富转基因甘蔗育种候选基因库,该研究结合蔗茅转录组数据,以蔗茅99 1无性系为试验材料,利用RT PCR技术克隆蔗茅MYB基因,并对其进行生物信息学分析及胁迫表达分析,以解析蔗茅的耐寒机理,为转基因甘蔗育种奠定理论基础。结果表明：(1)成功克隆得到一个蔗茅MYB基因,命名为EfMYB1基因(登录号ON586646)。(2)生物信息学分析表明,EfMYB1基因全长1 000 bp,ORF为759 bp,编码251个氨基酸;编码蛋白具有一个保守的SANT结构域,无跨膜结构和信号肽,有多个磷酸化位点;二级结构与三级结构主要以α螺旋和无规则卷曲为主;与南荻相似性最高,遗传距离最近。(3)qRT PCR分析结果发现,EfMYB1基因在蔗茅根和叶组织中的相对表达量随低温胁迫时间的持续而逐渐显著上调,并于胁迫72 h时达到最大值,而在茎中的表达则几乎没有变化;茉莉酸甲酯胁迫下,EfMYB1基因的相对表达量呈先升高后降低的趋势,且在处理6 h时达到最高值;脱落酸胁迫下EfMYB1基因的表达水平较0 h时极显著降低。研究认为,EfMYB1基因属于低温胁迫响应基因,可能参与蔗茅低温胁迫下的应答反应。     

水母雪莲花青素合成相关基因的克隆及表达

为研究高山植物水母雪莲对强紫外辐射的分子适应机制,采用RT PCR结合RACE技术从水母雪莲中克隆了参与调控花青素合成的相关基因(SmMYB1),并采用hi TAIL PCR方法扩增了该基因的启动子序列。结果表明：(1)序列分析显示,SmMYB1基因cDNA序列(GenBank 登录号 MT188353)全长1 011 bp,编码269个氨基酸,gDNA序列含有2个内含子和3个外显子。(2)生物信息学分析显示,SmMYB1基因编码蛋白和菊科MYB蛋白亲缘关系最近,含有保守的［DE］Lx(2)［RK］ x(3)Lx(6)Lx(3)R和ANDV两个模体,属于拟南芥MYB第六亚族。SmMYB1基因启动子(GenBank 登录号 MT188354)全长1 407 bp,含有多个光响应元件。(3)荧光定量分析发现,SmMYB1基因在根、茎、叶和花中均表达,且在花中的表达量最高;在紫外胁迫下,SmMYB1基因的表达量在4 h达到最高,随后逐渐降低。研究推测SmMYB1基因可能参与调控水母雪莲花青素的合成以及对紫外辐射的响应过程。     

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。     

烟草NtSKOR1基因的克隆及功能分析

该研究利用拟南芥AtSKOR蛋白序列,通过NCBI的Blast搜索获得烟草NtSKOR1基因CDS序列。设计全长CDS扩增引物,通过RT PCR方法从栽培烟草中克隆得到NtSKOR1基因,对其进行生物信息学、表达特性等分析,并利用CRISPR/Cas9技术获得NtSKOR1的敲除材料。结果表明：(1)NtSKOR1基因CDS由2 466 bp核苷酸组成,编码821个氨基酸,推测NtSKOR1蛋白等电点为6.36,分子量为94.21 kD。(2)NtSKOR1定位于细胞膜,有6个跨膜区,无信号肽序列;NtSKOR1蛋白含有孔形成区(P)及锚定蛋白区(ANK)等典型SKOR类蛋白功能域。(3)进化树分析表明,烟草NtSKOR1蛋白与番茄和马铃薯的SKOR蛋白遗传距离最近,与禾本科植物的SKOR蛋白遗传距离较远。(4)组织特异性表达分析表明,NtSKOR1基因主要在烟草根中表达,其表达模式与拟南芥一致;钾胁迫处理后,NtSKOR1基因呈先降低后升高再降低的表达模式。(5)CRISPR/Cas9敲除NtSKOR1基因,烟草叶片钾含量显著降低,表明NtSKOR1基因是控制烟叶钾离子的关键基因之一。该研究结果为解析烟草钾离子吸收转运的分子机制提供重要依据。     

蓖麻RcMsc2基因功能分析

G2/有丝分裂特异性细胞周期蛋白 2(G2/mitotic-specific cyclin-2,Msc2)作为高等植物应对逆境胁迫的关键调控蛋白,参与多个抗逆境胁迫的应答。为探究RcMsc2基因的功能,该研究从蓖麻叶片组织中成功克隆了RcMsc2,并利用生物信息学分析RcMsc2蛋白的结构和潜在功能,同时借助qRT-PCR方法分析RcMsc2基因的组织表达特性和非生物胁迫表达特性。结果表明:(1)RcMsc2基因位于蓖麻第5号染色体长臂,该基因的CDS(coding sequence)区是1 299 bp,编码432个氨基酸。(2)RcMsc2蛋白拥有细胞周期(cyclin)家族特征结构域,是一个不稳定酸性亲水蛋白,无跨膜域和信号肽,相对分子量为49.38 kD。(3)RcMsc2蛋白质的二级、三级结构以α-螺旋和无规则卷曲为主。(4)RcMsc2蛋白与麻风树和巴西橡胶树的CYCB2蛋白的序列同源性最高,且同被聚为Group Ⅱ。(5)35S-RcMsc2-GFP融合蛋白定位于细胞核。(6)RcMsc2基因在蓖麻的所有组织中均有表达且主要在根和茎中发挥作用; 非生物胁迫分析表明RcMsc2基因可以被脱落酸(abscisic acid, ABA)、盐、干旱和低温处理诱导表达,并且RcMsc2基因对低温胁迫的响应最敏感。综上表明,该研究较全面地分析了RcMsc2基因的结构特征、系统进化和表达模式,为揭示RcMsc2基因在蓖麻的生长发育和应答冷胁迫过程中的功能提供了理论参考。     

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.     

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.     

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。     

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.     

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.