春兰AGL6-3基因的克隆及实时定量表达分析

该研究以春兰(Cymbidium goeringii)正常花及其2枚侧瓣突变成唇瓣样的花瓣(简称:蝶花)为实验材料,采用RT-PCR结合RACE技术从春兰中分离出AGL6-3基因。序列分析表明,AGL6-3基因在春兰正常花和蝶花中序列相同,该基因含有1个720bp长的开放阅读框(ORF),共编码239个氨基酸。系统进化树进行分析表明,该基因属于MADS-box基因中AP1/AGL9组的AGL6同源基因,命名为CgAGL6-3(基因登录号为KU058679)。实时荧光定量表达分析表明,CgAGL6-3在春兰正常花和蝶花各花器官中表达存在差异。在正常春兰中CgAGL6-3基因在唇瓣中强烈表达,在主萼、侧萼及蕊柱中表达量较低,在侧瓣中则微乎其微;而在蝶花中CgAGL6-3基因在唇瓣中强表达,侧瓣中的表达量次之,在主萼、侧萼和蕊柱中的表达量相近且均较低。研究说明,CgAGL6-3基因可能在春兰蝶花侧瓣唇瓣化的过程中扮演重要角色。     

枸杞花药发育基因LbMS2的克隆及表达分析

MS2(Male sterily2)类基因编码脂肪酰基还原酶,参与了花药绒毡层中脂类代谢过程。该研究以宁夏枸杞(‘宁杞1号’)为试验材料,采用RACE方法从枸杞花药cDNA中获得枸杞LbMS2基因。结果显示,LbMS2基因开放阅读框为1 782bp,编码593个氨基酸,等电点为8.98;生物信息学分析显示,LbMS2蛋白定位于叶绿体;LbMS2蛋白序列与茄科植物矮牵牛、茸毛烟草、马铃薯、番茄以及甜辣椒中的MS2蛋白表现出较高的序列相似性;实时荧光定量PCR结果显示,LbMS2基因具有器官表达特异性,只在枸杞花器官中表达,并且在枸杞花药发育的四分体时期以及单核花粉时期表达量最高。研究表明,LbMS2基因是枸杞花器官发育过程中的重要基因。     

CygoSTK基因在普通春兰与奇花品种‘天彭牡丹''中的表达比较

为深入研究春兰(Cymbidium goeringii)与春兰奇花品种花器官发育调控的分子机制,该研究采用同源克隆的方法,分别从普通的春兰与春兰奇花品种‘天彭牡丹’的花芽中克隆得到1个cDNA长849 bp D类MADS-box基因CygoSTK(Genbank登录号为MH917912.1)。结果表明:该基因序列在两种春兰中高度一致,包含1个长705 bp的完整ORF,编码1个由234个氨基酸残基组成的STK进化系MADS-box转录因子;结构分析表明,CygoSTK转录因子包含1个高度保守的MADS结构域(MADS domain)(1～57)和1个次级保守的K结构域(91～172),其C末端的转录激活区含有两个高度保守的基序,即AGI基序和AGⅡ基序;进一步用qPCR检测CygoSTK基因在普通的春兰与春兰奇花品种‘天彭牡丹’不同花器官中的相对表达量发现,CygoSTK基因在普通的春兰和春兰‘天彭牡丹’子房中的表达量最高,显著高于该基因在相应品种其他花器官中的表达量(LSD,P0.05)。以上结果说明CygoSTK基因在功能上有很强的保守性,主要参与春兰子房的发育。     

向日葵MADS-Box基因HAM23-like克隆和表达分析

为了解MADS-box基因在向日葵(Helianthus annuus)花发育过程中的作用,采用RT-PCR技术克隆了1个MADS-box基因新成员HAM23-like,开放阅读框为831bp,编码276个氨基酸,相对分子量为30.52k D,理论等电点为9.42。系统发育分析表明,HAM23-like与拟南芥的AGL18聚于同一分支,具有较近的亲缘关系。qRT-PCR分析表明,HAM23-like基因在花和成熟果实(籽粒饱满期)中的表达量较高;HAM23-like在开花当天的雄蕊中的表达量最高;随着花的发育,HAM 23-like表达量逐渐升高,在开花后5 d (果实形成早期)达到最高表达水平。因此,推断HAM23-like基因可能与向日葵花器官后期发育和瘦果早期发育相关。     

棉花MADS框蛋白基因(GhMADS1)的克隆

作为转录因子,MADS框蛋白基因在植物花器官发育中有着重要的功能。为研究棉花花器官发育的分子机理,以棉花花器官突变体CHV1(cotton homeotic variant)和徐州142正常植株为材料,利用棉花EST数据库资料,通过EST序列整合,从陆地棉徐州142花蕾中克隆出一个MADS框蛋白的编码区段,GenBank登录号为AF538965。该片段(GhMADS1)长713bp,包含一个711bp的开放阅读框,推导的氨基酸序列(236个氨基酸)与葡萄、烟草、矮牵牛、拟南芥和金鱼草等的AGL2组MADS框蛋白有很高的序列相似性。系统进化分析同样将GhMADS1基因归人AGt2组MADS框蛋白。RT-PCR分析显示,该基因在陆地棉的花瓣、雄蕊、胚珠和纤维中表达,特别是在花瓣中表达量最高,而在根、茎、叶等营养器官和棉花同源异型突变体CHV1(所有花器官均变为苞叶状叶性器官)的变异花蕾中不表达。这些结果说明GhMADS1基因可能在棉花花器官发育中有着重要的功能。     

三倍体枇杷花期调控基因EjAGL6的表达特性和功能分析

以三倍体枇杷(Eriobotrya japonica) ‘华玉无核1号’的花芽为材料,采用基因克隆技术获得EjAGL6基因,分析其序列、亚细胞定位特性以及在二倍体和三倍体枇杷早晚花品种中的表达水平。采用花序浸染转化拟南芥,并利用实时荧光定量PCR分析转基因拟南芥植株的EjAGL6基因表达量,进一步观察野生型与EjAGL6转基因拟南芥的表型差异,分析EjAGL6基因的功能,为解析EjAGL6基因参与三倍体枇杷花期调控机制提供理论依据。结果显示：(1)成功获得MADS box基因EjAGL6;该基因的编码区序列(CDS)为732 bp,编码243个氨基酸,分子质量为27.88 kD,等电点为 9.05,脂溶指数为 79.05;系统进化树分析表明,枇杷EjAGL6与苹果MdAGL6蛋白质的相似性较高,聚在同一分支。(2)蛋白序列比对发现,EjAGL6的M区有57个氨基酸,I区有30个氨基酸,K区有82个氨基酸,C区有74个氨基酸,其中C区包含高度保守的AGL6基序Ⅰ和AGL6基序Ⅱ。(3)亚细胞定位分析表明,EjAGL6蛋白定位在细胞核,具有典型的MADS box转录因子亚细胞定位特性。(4)实时荧光定量PCR分析表明,EjAGL6基因在二倍体和三倍体枇杷早、晚花品种中均有表达,主要集中于小花分化期(S6)、花蕾露白期(S7)和盛花期(S8),且EjAGL6基因在二倍体和三倍体早花品种中的花蕾露白期的表达量均较高。(5) 转基因拟南芥株系的EjAGL6基因表达量显著高于野生型拟南芥;转EjAGL6基因植株表型观察显示,EjAGL6基因在拟南芥中过量表达能够使转EjAGL6基因拟南芥的开花时间提前1周左右。研究认为,EjAGL6基因可促使枇杷开花时间提前,推测EjAGL6基因在花蕾露白期发挥调控花期的关键作用。     

桂花查尔酮异构酶OfCHI基因的克隆与表达分析

查尔酮异构酶CHI是花青素苷合成途径中的关键酶。为了解桂花花青苷的合成机理,该研究对3个桂花品种的花青苷含量进行了测定。结果显示:(1)‘橙红丹桂’花中的花青苷含量最高,‘金桂’和‘早银桂’中花青苷含量较低。(2)利用RACE和RT-PCR方法获得了桂花查尔酮异构酶基因(OfCHI)的全长cDNA序列1 069bp,该基因编码248个氨基酸,相对分子量为26.85kD,等电点为6.34。(3)多重序列比对显示,OfCHI与金花茶CnCHI、忍冬LjCHI、石榴PgCHI的相似性分别为68.13%、65.86%和63.53%,氨基酸序列中含有CHI蛋白的活性位点Thr47、Tyr108、Met115以及Ser192;系统进化树分析表明,OfCHI与其他物种起源相同,而与油橄榄OeCHI亲缘关系最近。(4)利用qRT-PCR对不同桂花品种、不同组织中OfCHI的表达量检测结果显示,OfCHI在‘橙红丹桂’花中表达量最高,在‘金桂’和‘早银桂’中表达量较低;OfCHI在‘橙红丹桂’、‘金桂’和‘早银桂’的花、茎、叶中均有表达,且表达趋势相同,均为叶中表达量最高。该研究为揭示桂花青素苷的合成机理奠定了理论基础,并为培育不同花色的桂花新品种提供了基因专利。     

春兰CgSEP3基因的克隆和表达分析

该研究采用RT-PCR和RACE技术从春兰(Cymbidium goeringii)中分离到1个SEPALLATA3(SEP3)基因。序列分析表明,该基因含有1个732bp的开放阅读框(ORF),共编码243个氨基酸。系统进化树分析显示,该基因是MADS-box基因家族AP1/AGL9组SEP的同源基因,其编码蛋白与其它植物SEP3类蛋白具有较高的一致性,命名为CgSEP3(登录号为KF924272)。实时荧光定量分析表明,CgSEP3在春兰花器官中均有表达,其中在唇瓣、侧瓣和萼片中的表达量较高,在子房和蕊柱中的表达量较低;而且CgSEP3在花发育各个时期都有表达,在1~2cm的花芽中表达量最高,在盛开的花中的表达量最低。研究认为,CgSEP3基因可能在春兰花瓣和萼片的形成过程中具有重要作用。     

大豆GmAP1基因的功能初探

以大豆天隆1号为材料,根据Glycine max Wm82.a2.v1中大豆基因组的预测序列,克隆出一个AP1同源基因,命名为GmAP1。该基因编码区CDS长度为711 bp,编码一个含236个氨基酸的蛋白质。对蛋白质序列的结构分析结果显示GmAP1蛋白符合MADS-box基因家族特征。为了初步分析GmAP1的功能,利用实时定量RT-PCR分析GmAP1在不同花器官中表达,并且在拟南芥中过表达该基因。在不同花器官中,GmAP1表达量不同,在花萼中表达量较高;花瓣次之。GmAP1过表达植株表现出早花、植株矮小以及花器官(花瓣、雄蕊和雌蕊)数量增多等表型。结果表明,大豆的GmAP1是一个功能保守的AP1基因,在花的发生和花器官发育中起着重要作用。     

百合LbKCS5和LbKCS17基因的克隆与表达分析

β 酮脂酰辅酶A合成酶(KCS)是超长饱和脂肪酸链(VLCFAs)生物合成中的限速酶,该研究以百合(Lilium brownii var. viridulum Baker.)‘黄天霸’cDNA为模版, 采用RT PCR方法克隆了LbKCS5和LbKCS17基因的CDS序列,并进行序列分析,采用qRT PCR方法分析其基因表达以及胁迫诱导特征。结果表明：(1)LbKCS5和LbKCS17分别包含689和1 238 bp完全阅读框(ORF), 编码186和291个氨基酸;进化分析显示, LbKCS5与油棕KCS5、LbKCS17与莲KCS17序列相似性最高,分别为82.61%和77.62%。(2)基因表达分析结果显示, LbKCS5和LbKCS17 在百合营养器官中均有表达,二者在叶中表达量最高,在根中最少;在花器官中,LbKCS5的表达量在雄蕊中最高,LbKCS17在花药中最高,在花瓣中二者的表达量最低;在花蕾生长发育过程中,花瓣中2个基因的表达均先升高后下降,并于黄蕾期达到峰值;叶中LbKCS5表达量在黄蕾期达到最高,而LbKCS17在叶中的表达整体较低。(3)失水胁迫能提高这2个基因的转录表达,低温诱导LbKCS5表达量的增加,但却降低了LbKCS17的表达。研究表明,LbKCS5和LbKCS17在百合不同器官中均有表达,且均响应失水和低温胁迫,这为研究百合耐失水和低温胁迫以及新品种选育提供了理论支持。     

Coccystes undoxylophus

Ohne Zusammenfassung     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Aquila pennata undminuta

Ohne Zusammenfassung     

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.     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

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.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.