水芹类黄酮3'-羟化酶基因的克隆与表达特性分析

类黄酮3'-羟化酶(flavonoid 3′-hydroxylase,F3'H)属于细胞色素P450家族(cytochromeP450,CYP450),在植物主要成色物质花青素的合成中发挥重要作用。本实验以‘八卦洲水芹’以及紫色叶柄突变型水芹为实验材料,利用RT-PCR方法,从紫色叶柄突变型水芹的cDNA中克隆得到编码类黄酮3'-羟化酶的基因,命名为OjF3'H1。序列分析显示,OjF3'H1基因全长1575bp,共编码524个氨基酸。OjF3'H1蛋白相对分子质量为58292.59,理论等电点为6.77。系统进化分析显示,OjF3'H1蛋白具有高度保守性,与同属伞形科的胡萝卜F3'H1进化关系最近。OjF3'H1编码的蛋白属于疏水蛋白,无序化比例为5.53%,空间结构主要由α-螺旋和β-折叠组成。实时定量PCR分析显示,OjF3'H1在不同品种水芹茎中相对表达量有明显差异,紫色叶柄突变型水芹中OjF3'H1的表达量明显比‘八卦洲水芹’中高。     

芹菜品种‘六合黄心芹’ AgCCoAOMT基因的克隆及表达特性分析

根据伞形科( Apiaceae)植物芹菜( Apium graveolens Linn.)的转录组数据库,从芹菜品种‘六合黄心芹’(‘Liuhe Huangxinqin’)叶片总RNA中克隆获得咖啡碱-辅酶A-O-甲基转移酶基因,命名为AgCCoAOMT基因。序列分析结果表明：AgCCoAOMT基因包含1个长度726 bp的开放阅读框( ORF),编码241个氨基酸;该基因编码的蛋白质为AgCCoAOMT,其理论相对分子质量为27010,理论等电点pI 5.35;在AgCCoAOMT蛋白中,酸性氨基酸所占比例高于碱性氨基酸,脂肪族氨基酸所占比例约为芳香族氨基酸的3倍;该蛋白属于疏水性蛋白,并含有1个保守的AdoMet MTases结构域,说明AgCCoAOMT蛋白属于AdoMet MTases超级家族;AgCCoAOMT蛋白的三级结构包含多个a-螺旋和β-折叠,与紫花苜蓿( Medicago sativa Linn.) CCoAOMT蛋白三级结构的一致性为84.58%。多重比对和系统树分析结果表明：AgCCoAOMT蛋白有较高的保守性;该蛋白与同科植物大阿米芹( Ammi majus Linn.)和欧芹﹝Petroselinum crispum ( Mill.) Nyman ex A. W. Hill〕CCoAOMT蛋白的进化关系最近,与睡莲科( Nymphaeaceae)植物莲( Nelumbo nucifera Gaertn.) CCoAOMT蛋白的进化关系较近。 qRT-PCR检测结果表明：AgCCoAOMT基因能够在‘六合黄心芹’的根、茎、叶柄和叶片中表达,且在叶片中的相对表达量极显著(P<0.01)高于其他组织,说明该基因的表达具有明显的组织特异性;不同生长发育阶段该基因在叶片中的相对表达量有明显差异,其在商品期(播种后第65天)的相对表达量极显著高于幼苗期(播种后第25天)和生长旺盛期(播种后第45天)。研究结果显示：AgCCoAOMT基因与‘六合黄心芹’叶的老化和木质素含量升高有关,且在进化过程中具有较高的保守性。     

胡萝卜肉桂醇脱氢酶基因的克隆及其对非生物胁迫的响应

该实验采用RT PCR技术,从胡萝卜‘黑田五寸’中克隆获得了编码肉桂醇脱氢酶（Cinnamyl alcohol dehydrogenase, CAD）的基因DcCAD。DcCAD序列长1 074 bp,编码357个氨基酸。亲水/疏水性分析表明,DcCAD属于亲水性蛋白。系统进化分析显示,DcCAD与番茄CAD（XP_010314515.1）亲缘关系最近。荧光定量PCR结果显示,DcCAD基因在胡萝卜根、叶片和叶柄中的表达量差异显著,其相对表达量为叶片＞根＞叶柄。DcCAD基因对高温（38 ℃）、低温（4 ℃）、干旱（20% PEG）和盐（0.2 mol·L^-1 NaCl）胁迫均有响应,尤其对高温胁迫和低温胁迫响应明显,而且高温处理后1 h和低温处理后2 h表达量最高。研究推测,DcCAD基因对胡萝卜抗逆性具有一定的作用。     

苋菜甜菜红素合成相关基因AmMYB1的克隆及表达分析

应用RACE技术,从‘大红’苋菜中克隆到1条MYB基因cDNA全长序列,命名为AmMYB1(登录号为KU557504)。AmMYB1基因开放阅读框为723bp,可编码240个氨基酸。其基因组序列与cDNA比对后显示,AmMYB1基因含有1个内含子。生物信息学分析表明,AmMYB1具有2个连续的MYB结构域,是一个典型的R2R3-MYB;同源分析显示,该基因编码的氨基酸序列与甜菜红素相关BvMYB1的一致性最高,达到54%。亚细胞定位结果显示,AmMYB1蛋白定位于细胞核。实时荧光定量PCR分析表明,AmMYB1基因在‘大红’苋菜叶片红色部位的表达量高于绿色部位;在甜菜红素含量高的叶和茎中表达量明显高于根;在光照条件下表达量高于遮光处理;在红叶品种中的表达量高于绿叶品种。研究结果表明,AmMYB1基因可能是苋菜甜菜红素合成途径中重要的正调控因子。     

文心兰FT同源基因的克隆及其表达分析

FT(Flowering Locus T)及其同源基因被认为是植物开花、花发育相关的重要基因。为了深入研究FT同源基因的功能以及文心兰开花的分子机理,该研究以文心兰品种‘金辉’为试验材料,基于转录组测序结果,克隆获得‘金辉’FT同源基因,命名为OnFT(GenBank登录号为MK967676)。OnFT基因编码区长度为537 bp,共编码178个氨基酸;生物信息学分析表明,该蛋白质分子量约为19.99 kD,可能是一种亲水性不稳定蛋白质,属于PEBP家族蛋白;基于FT的系统进化分析表明,文心兰与同科植物桃红蝴蝶兰的亲缘关系较近。qRT-PCR分析结果显示,OnFT在文心兰不同组织的表达差异较大,在花中表达量最高,在根中几乎不表达;OnFT基因在幼嫩花芽中的表达量较低,并且在花的成熟过程中逐渐增加;OnFT基因在叶片和假鳞茎中的表达量随成熟度的增长均呈先上升后降低的变化趋势,在中苗期的叶片和抽芽期的假鳞茎中表达量较高。     

中国古老月季‘月月粉’FT/TFL1基因的鉴定与表达分析

该研究以中国古老月季品种‘月月粉’(Rosa chinensis‘Old Blush’)为试材,根据拟南芥和草莓FT/TFL1基因家族成员的保守结构域设计引物,对‘月月粉’基因组DNA进行克隆和测序,结合数据库序列和双单倍体基因组信息进行序列比对,结果共获得了8条月季的FT/TFL1基因家族序列,且8条序列分别注释为2个FT基因(RcFT1和RcFT2)、1个BFT基因(RcBFT)、1个ATC基因(RcATC)、2个MFT基因(RcMFT1和RcMFT2)和2个PEBP基因(PEBP1和PEBP2);进化树分析结果将这8个基因分为3个亚组;结构分析显示8个基因的内含子为1～5个不等。qRT-PCR分析显示,RcFT1、RcPEBP、RcMFT1、RcMFT2和RcBFT在‘月月粉’的叶片中表达量最高,茎尖中次之;RcFT2在茎尖中的表达最高,叶片中次之;RcATC在茎尖中的表达量高于其他组织;除RcPEBP在根组织中有表达外,其他基因在根组织中几乎没有检测到。进一步表达分析显示,RcFT1和RcFT2在‘无刺光叶蔷薇’(R.wichuriana‘Basye’s Thornless')生殖生长期的叶片、茎尖中的表达量显著高于营养生长期。研究推测,具有连续开花性状的中国古老月季‘月月粉’可能具有多个开花时间(FT)基因位点,FT有可能可以作为月季生殖转换的标记基因。     

‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化

为探究多花水仙ACS基因的序列特征及功能,以‘云香’水仙盛花期花瓣为试验材料,根据‘云香’水仙花朵转录组数据信息,通过RT-PCR方法克隆出1个ACS基因,命名为NtACS1(GenBank KX082936);NtACS1开放阅读框(ORF)长度为552bp,编码183个氨基酸。编码蛋白质分子量约为20.6KDa,理论等电点为6.30,不稳定系数为65.49,属于不稳定的疏水性蛋白。通过qRT-PCR对‘云香’水仙不同时期花瓣和副冠中的NtACS1基因进行了表达分析,得到与‘云香’水仙花朵转录组数据中相同的结果:NtACS1基因在‘云香’水仙花瓣和副冠中的表达都是随着花衰老过程呈现逐渐下降的趋势,且NtACS1基因在花瓣和副冠中的表达峰值都在花苞期,表明NtACS1基因编码的蛋白是在乙烯生物合成途径的系统1发挥催化作用的ACC合成酶。成功构建了NtACS1基因的正义植物表达载体,并通过农杆菌介导法获得8株转基因烟草,PCR和RT-PCR检测显示其中有6株为阳性植株,初步证实NtACS1基因已导入烟草基因组中且在烟草中已表达。该研究结果为进一步分析NtACS1基因的功能和后续转化水仙延长其花期研究奠定了基础。     

小麦TaWRKYⅢ-A37和TaWRKYⅡc-D2基因的克隆与表达分析

为了探究小麦(Triticum aestivum L.)WRKY基因的功能,采用同源克隆的方法,从小麦品种‘科农199’中克隆得到2个WRKY基因,分别命名为TaWRKYⅢ-A37和TaWRKYⅡc-D2,并对其进行生物信息学分析和不同逆境胁迫下的表达分析。生物信息学分析显示,TaWRKYⅢ-A37和TaWRKYⅡc-D2基因都含有2个内含子和3个外显子,分别编码206和138个氨基酸,编码蛋白都属于亲水性不稳定非分泌型的核蛋白。系统进化分析表明,TaWRKYⅢ-A37蛋白与其两个同源拷贝的亲缘关系最近,而TaWRKYⅡc-D2蛋白与粗山羊草亲缘关系最近。qRT-PCR结果表明,TaWRKYⅢ-A37和TaWRKYⅡc-D2基因在小麦根、茎和叶中均有表达,前者在根中表达量最高,后者在叶中表达量最高,二者均在茎中低表达;在苗期TaWRKYⅢ-A37基因受到PEG、H_2O_2和ABA胁迫后表达上调,NaCl处理后4～8 h内表达下调且低于对照表达水平,而且在灌浆期受到PEG、NaCl、H_2O_2和ABA胁迫后表达均上调;苗期TaWRKYⅡc-D2基因在NaCl、H_2O_2和ABA胁迫后表达下调,PEG处理2 h时表达上调且高于对照表达水平,并且在灌浆期经PEG和NaCl胁迫后表达下调,受H_2O_2和ABA胁迫后表达上调。该研究结果为深入探究TaWRKYⅢ-A37和TaWRKYⅡc-D2基因的抗逆功能奠定了理论基础。     

玉米生物钟基因ZmPRR1-2的克隆及表达分析

为探究玉米生物钟基因ZmPRR1-2的功能及表达特性,解析玉米光周期途径调控开花的机理,该研究以玉米骨干自交系‘黄早4’为材料,克隆ZmPRR1-2基因的cDNA序列并进行生物信息学分析;利用qRT-PCR技术对该基因进行组织特异性表达分析和48 h的昼夜节律表达分析。结果表明：(1)成功克隆获得ZmPRR1-2基因的编码区全长1 554 bp,编码517个氨基酸,编码的蛋白属于PRR基因家族,含有1个REC结构域和1个CCT结构域,多序列比对和系统进化分析显示ZmPRR1-2基因在禾本科植物中高度保守;ZmPRR1-2蛋白属亲水性蛋白,不包含跨膜结构域和信号肽。(2)ZmPRR1-2基因在玉米叶片中的表达量最高,显著高于其他7个组织,表明该基因主要在叶片中发挥功能,而在果穗和花丝中表达量相对较低,且显著低于雄穗中的表达量。(3)昼夜节律表达分析显示,在短日照条件下,ZmPRR1-2基因的表达量于光照3 h后开始逐渐上升,在光照结束后3 h时达到表达高峰;在长日照条件下,于光照6 h后ZmPRR1-2基因的表达量才开始逐渐上升,且在光照结束时达到表达高峰。研究认为,ZmPRR1-2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.     

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.     

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.     

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.     

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.     

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。     

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.