独行菜种子bHLH类转录因子基因家族及幼苗laICE1表达对冷胁迫的响应

该研究在转录组数据基础上,以独行菜(Lepidium apetalum Willd.)种子耐受和不能耐受低温萌发的2组样本为材料,对其bHLH转录因子家族成员进行分析,并对该家族成员表达差异极显著的laICE1基因进行克隆、序列分析、结构预测,以探讨独行菜幼苗中laICE1基因表达对低温胁迫的响应特征。结果表明:(1)萌发中的独行菜种子,至少有83个bHLH转录因子序列表达,相对于低温萌发停滞组,在低温萌发耐受组的独行菜种子中,有10个下调、13个上调、60个表达差异不显著。其中表达量极显著下调的序列c20009_g1具有1 503bp开放阅读框,GO注释到ICE1基因,该基因命名为laICE1。(2)laICE1基因编码500aa,蛋白分子量为54 635.19kD,理论等电点为5.45,分子式为C2364H3742N688O758S22;该蛋白具有保守结构域bHLH。(3)定量分析表明,laICE1基因在非低温胁迫的独行菜种子中的表达量显著低于一直处于低温胁迫中不能进行萌发的独行菜种子,这与转录组数据库中该序列表达情况一致;而laICE1基因在独行菜幼苗期经低温处理后,其表达量显著上调,表明laICE1基因可能在独行菜幼苗耐受低温生长中具有一定的作用。     

独行菜LaAP2基因克隆、生物信息学分析及表达分析

AP2/ERF是广泛存在于植物中一类重要的转录因子,调控一些参与非生物胁迫相关基因的表达,帮助植物提高逆境胁迫能力。为了深入探讨LaAP2在独行菜耐受低温萌发及幼苗耐受低温生长中的功能,该研究基于前期对独行菜(Lepidium apetalum)转录组数据库分析,克隆获得一个显著上调表达的AP2/ERF家族序列LaAP2。该基因cDNA全长为1 005 bp,编码氨基酸序列包含一个AP2和一个B3结构域,属于AP2/ERF转录因子RAV亚家族。推定的LaAP2蛋白分子量为37.744 67 kD,等电点为9.49。该蛋白氨基酸序列同亚麻荠、拟南芥、油菜等物种显示出较高同源性,系统进化分析结果表明与拟南芥亲缘关系较近。氨基酸序列分析预测表明,LaAP2基因所编码的蛋白不具备信号肽区段,无跨膜区,不属于分泌蛋白,可能为亲水性蛋白;定位于细胞质的可能性为56.5%,定位于细胞核的可能性为21.7%;其主要二级结构元件为无规则卷曲、延伸链、α-螺旋。Real-time PCR分析独行菜幼苗中LaAP2在低温4℃处理下的表达,显示LaAP2表达受低温胁迫呈先下降后升高趋势。这表明LaAP2在独行菜幼苗抵抗低温胁迫中起调控作用。     

拟南芥ABI5基因对BR胁迫响应及其对下胚轴生长的调节作用

植物脱落酸不敏感蛋白5 (abscisic acid-insensitive 5,ABI5)是种子中大量表达的碱性亮氨酸拉链类型(basic leucine zipper,b ZIP)转录因子,在调节种子萌发和幼苗早期生长的脱落酸(abscisic acid,ABA)信号中起着核心作用。油菜素内酯(brassinosteroid,BR)是一种新型植物内源激素,具有调节植物生长发育和逆境胁迫响应等诸多生理功能。近期研究发现,油菜素内酯胁迫条件下,BR信号通路中BIN2 (BRASSINOSTEROID INSENSITIVE2)和BES1 (BRI1-EMS-SUPPRESSOR 1)通过抑制ABI5表达,促进拟南芥(Arabidopsis thaliana)种子萌发。为进一步探究BR胁迫下ABI5功能,本研究分析了种子萌发期ABI5表达特性,鉴定出拟南芥ABI5基因缺失突变体abi5-1并对BR胁迫下其功能进行解析。结果表明:ABI5在拟南芥干种子中大量表达并响应萌发期BR胁迫;正常条件下,abi5-1与野生型幼苗下胚轴无明显差异;BR胁迫下,abi5-1幼苗下胚轴明显长于野生型。本研究结果揭示了ABI5调控BR胁迫下拟南芥下胚轴生长,为深入了解ABI5调节植物发育的分子机制提供了依据。     

独行菜冷上调基因LaNHR2B的低温耐受性研究

新疆北部早春短命植物独行菜幼苗期能够在早春的低温条件下生长,具有良好的耐受低温胁迫的能力。前期研究获得了独行菜幼苗冷诱导上调表达基因片段,通过同源克隆获得该基因的全长cDNA序列（LaNHR2B）,运用生物信息学软件预测分析该基因编码蛋白的性质及其构象,采用荧光定量RT PCR技术分析其表达量与幼苗生长阶段、冷诱导处理以及外源ABA处理间的关系,通过转化拟南芥研究过表达该基因对植物幼苗低温耐受性的影响。结果表明：（1）LaNHR2B基因全长1 035 bp,编码344个氨基酸,蛋白质分子质量为85 791.16 kD,理论等电点为5.06,分子式为C₃₁₂₉H₅₂₂₅N₁₀₃₅O₁₃₀₇S₂₃₅;其蛋白主要由丙氨酸、苏氨酸、甘氨酸及半胱氨酸组成,具有3个跨膜结构,功能未知;在十字花科植物中保守性强,其他科的植物中未见同源基因。（2）LaNHR2B受4 ℃低温诱导显著上调表达,但随幼苗生长受低温诱导上调表达有所降低,与幼苗随发育耐受低温能力下降变化一致。（3）外源ABA可诱导LaNHR2B表达上调;过表达拟南芥幼苗低温耐受性明显增强。该研究初步证明,LaNHR2B表达量与独行菜幼苗耐受低温密切相关,可能是独行菜幼苗经过冷驯化后能够增强植株抗寒能力的功能性基因。这为进一步开发利用该基因进行油菜等作物抗寒育种提供理论依据。     

隐花素在生长素调控拟南芥下胚轴伸长中的作用分析

以拟南芥野生型(Col-4)和隐花素双突变体cry1cry2为材料,研究不同光照条件下不同浓度吲哚乙酸(IAA)和IAA极性运输抑制剂氨基酞氨酸(NPA)对幼苗下胚轴伸长的影响。结果显示,低浓度IAA(10-7mol/L)可促进连续白光和红光下cry1cry2幼苗下胚轴伸长,而连续蓝光下cry1cry2下胚轴的伸长则受到抑制。蓝光下相同浓度的NPA对cry1cry2幼苗下胚轴伸长的抑制程度比野生型要小。RT-PCR分析结果显示,瞬时蓝光处理时IAA合成关键酶基因IGPS以及生长素应答基因IAA1和IAA5在cry1cry2突变体中的转录水平比野生型中要高。这表明隐花素可能部分通过调节IAA合成和/或IAA极性运输,介导蓝光调控拟南芥下胚轴的伸长。     

短命植物独行菜种子萌发过程对低温的耐受特性

以独行菜（Lepidium apetalum）为材料,研究了其种子在萌发过程中耐受低温的特性,并对耐受低温的机制作了初步的探讨。结果表明：1.萌发至I、II、III期的独行菜种子经过冷诱导处理后,对零下低温-5℃、-10℃胁迫具有较强的耐受性,这种耐受性随萌发发展相对有所降低。2.独行菜种子萌发过程中胚根生长速度明显受到低温抑制,但也仍然能够生长。3.适当时间的低温层积能使独行菜萌发势显著提高,对终萌发率影响不大;过长时间的低温层积会使独行菜种子萌发势和最终萌发率降低。4.独行菜种子在4℃条件下不能萌发,但4℃层积2d的种子经25℃处理6h以上后,可耐受4℃低温而萌发,且萌发率随25℃处理时间增加而升高。如果25℃处理时间少于5h则不能在4℃低温下萌发。经4℃层积10d、再经25℃萌发处理1h的独行菜种子就能在4℃低温下萌发。综合分析认为独行菜种子不能耐受4℃低温萌发,原因可能是在露白前存在一个关键的生理阶段,在4℃胁迫逆境中不能越过这个阶段,该阶段之前与之后的萌发过程都能耐受4℃低温,因此对低温胁迫有良好的耐受性。这为探索早春短命植物耐受低温萌发的机制提供了新的思路。     

Leukamenin E调节拟南芥幼苗生长发育的模式及其机制

研究对映-贝壳杉烷型二萜化合物Leukamenin E对拟南芥种子萌发、下胚轴伸长以及根生长发育的作用模式,并探讨植物激素生长素和乙烯可能介导Leukamenin E影响拟南芥主根生长、侧根和根毛发育的初步机制。结果表明:Leukamenin E浓度在10~160μmol·L~(-1)范围对拟南芥种子的萌发率无显著影响,但高浓度Leukamenin E(80~160μmol·L-1)显著抑制种子的萌发速率。Leukamenin E对拟南芥幼苗根生长的抑制作用明显高于对下胚轴的抑制效应。进一步研究表明,Leukamenin E通过阻滞根尖细胞有丝分裂和细胞伸长进而抑制主根的生长,并能促进侧根提前发生并影响其形成数量,同时减少根毛密度及降低根毛长度。Leukamenin E联合乙烯利(乙烯释放剂)处理可阻止乙烯利单独使用对拟南芥幼苗根毛生长的促进作用,与Ag+(乙烯竞争抑制剂)联合乙烯利的作用效果相一致,表明Leukamenin E可能通过干扰根细胞乙烯途径而抑制根毛发育。流动注射化学发光分析和酶联免疫检测的结果发现,Leukamenin E显著上调拟南芥幼苗根组织中生长素(IAA)水平,表明生长素可能作为主要因子介导了Leukamenin E对拟南芥幼苗根生长发育的调节作用。     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

两种独行菜种子萌发生理特性研究

以两种在新疆具有代表性的十字花科独行菜属早春短命植物抱茎独行菜（Lepidium perfoliatum Linnaeus）和独行菜（Lepi-dium apetalum Willd）为材料,研究（光照、温度、外源激素ABA处理）对其种子萌发的影响。结果：两种独行菜种子萌发对光照不敏感;抱茎独行菜种子萌发的温度范围广,在0℃～25℃之间都可萌发,而独行菜种子则在低温下（0℃、4℃）不萌发,其萌发温度范围为10℃～25℃。两种独行菜种子对低温胁迫表现出不同的耐受性;0℃、4℃下,在培养基中加入不同浓度的ABA,对抱茎独行菜种子萌发有抑制作用。     

独行菜LaMCT基因的克隆、序列分析与原核表达

强心苷作为药用植物独行菜( Lepidium apetalum)的活性成分,其化学和药理学研究已有良好的基础,但其生物合成途径目前仍不清楚。该研究以独行菜幼苗为材料,通过分析独行菜转录组数据,设计特异性引物,PCR扩增得到了强心苷生物合成MEP途径的关键酶2-C-甲基赤藓醇-4-磷酸胞苷酰转移酶( MCT)基因的开放阅读框( ORF),命名为LaMCT( Genbank注册号KT832554),并进行序列分析和原核表达。序列分析结果表明：LaMCT基因ORF全长为912 bp,编码304个氨基酸。亚细胞定位和保守结构域分析结果表明：LaMCT蛋白位于叶绿体中,不含信号肽,没有跨膜区,含有类异戊二烯合成酶保守结构域( isoprenoid synthase domain)。系统进化树结果表明：LaMCT蛋白与拟南芥的MCT蛋白具有94％的序列相似性,亲缘关系较近。通过构建pET-32a-LaMCT原核表达载体,成功在大肠杆菌BL21( DE3)菌株中诱导表达LaMCT重组蛋白,并得到了纯化的LaMCT重组蛋白。该研究首次从独行菜中克隆了LaMCT基因,建立其稳定的原核表达体系,为LaMCT蛋白抗体的制备以及研究LaMCT基因在独行菜强心苷类化合物生物合成途径中的功能奠定了基础。     

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.     

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。     

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.     

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.     

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.