龙眼体胚发生过程中DlAGO4基因的克隆及表达分析

该研究基于龙眼基因组数据库,采用RT-PCR技术,以‘红核子’品种龙眼松散型胚性愈伤组织cDNA为模板,进行龙眼胚性愈伤组织DlAGO4基因的克隆和生物信息学分析,并采用实时荧光定量技术分析其在龙眼体细胞胚胎发生不同阶段、不同组织部位、激素和非生物胁迫处理以及5-氮胞苷(5-azac)处理的表达模式。结果表明:(1)DlAGO4基因cDNA全长为3 425 bp,包含开放阅读框长度为2 781 bp,编码926个氨基酸。(2)生物信息学分析表明,DlAGO4蛋白为碱性亲水非分泌蛋白,含有AGO经典的保守结构域PAZ和Piwi,与克莱门柚CcAGO4的同源性最近,含有85个磷酸化位点和2个糖基化位点;亚细胞定位预测其最可能定位于细胞核;MicroRNA预测显示,DlAGO4基因受到4个miRNA靶向调控。(3)qRT-PCR结果表明,DlAGO4在龙眼球形胚阶段和种子中相对表达量最高;激动素、水杨酸、NaCl、甘露醇、PEG-4000和ABA处理均能促进DlAGO4基因的表达,而2,4-D和MeJA处理抑制其表达;不同浓度的5-azac处理1 d和3 d抑制DlAGO4的表达,但从处理第6天开始该基因呈上调表达,并于处理第12天相对表达量最高。研究认为,DlAGO4基因可能参与龙眼球形胚和种子的转录调控,且可能参与KT、SA的激素信号转导途径和NaCl、甘露醇、PEG-4000、ABA的逆境胁迫响应途径以及DNA甲基化调控机制。     

建鲤两种Δ6脂肪酸去饱和酶基因克隆与表达

利用逆转录PCR和RACE技术获得建鲤2种Δ6脂肪酸去饱和酶（FADs6-a,FADs6-b）的全长cDNA序列. FADs6-a基因的cDNA总长为1 966 bp,开放阅读框为1 335 bp,编码444个氨基酸;FADs6-b基因的cDNA总长为1 931 bp,开放阅读框为1 335 bp,编码444个氨基酸.FADs6-a和FADs6-b基因都包括N端细胞色素b5结构域、3个富含组氨酸的结构域和2个推测的跨膜区,具有典型的Δ6脂肪酸去饱和酶结构特点.氨基酸同源性分析显示,建鲤FADs6-a和FADs6-b与斑马鱼的相似性较高,而与海水鱼类的相似性较低,与人类FADs6的相似性高于与FADs5的相似性.通过实时荧光定量PCR(RT-qPCR)检测该基因在建鲤幼鱼不同组织中的表达量, 发现2种Δ6 脂肪酸去饱和酶基因在建鲤肝脏的表达量最高,其次是肠、脑、肌肉、心和肾,而前肠高于后肠,FADs6-a的表达量高于FADs6-b.得到的结论是,建鲤具有2种类型的合成高度不饱和脂肪酸(HUFA)的关键酶-Δ6脂肪酸去饱和酶,在肝脏和肠道中的含量较多,且FADs6-a和FADs6-b在结构和组织的表达方面都存在差异,这为进一步研究建鲤HUFA的合成途径及调控机理奠定了基础.     

龙眼体细胞胚胎发生过程中DlWUS的克隆与表达分析

以龙眼‘红核子’LC2悬浮细胞系诱导的胚性愈伤组织为基本材料,按照龙眼体细胞胚胎同步化方法诱导获得龙眼体胚不同阶段材料,并以龙眼体细胞胚胎发生不同阶段混合材料作为试验材料,采用RT-PCR结合RACE技术分离并克隆龙眼中编码同源异型结构域蛋白的转录因子WUSCHEL(简称DlWUS)的cDNA全长及DNA序列,并进行序列分析与表达分析。结果表明:DlWUS的cDNA全长1 110bp,开放阅读框(ORF)858bp,共编码285个氨基酸(GenBank登录号为KM017506),DlWUS的DNA包含2个内含子。序列分析表明,DlWUS是一个不稳定的亲水蛋白,不含信号肽,亚细胞定位于细胞核,具跨膜结构和Homeodomain超级家族的保守结构域以及WUS转录因子家族特有的WUS box和EAR-like结构域,推测该目的基因确实为WUS转录因子。系统进化分析显示,龙眼DlWUS与脐橙WUS归为一个分支,亲缘关系较近。实时荧光定量PCR分析结果表明,在龙眼体细胞胚胎发生整个过程中,DlWUS均有表达,但仅在球形胚时期表达量较高,说明DlWUS可能主要在球形胚阶段发挥作用,并且在一定浓度范围内,外源施加IAA和GA3能够促进DlWUS基因的表达,而外源施加SA则抑制DlWUS基因的表达。     

丹参多酚氧化酶基因的克隆及表达分析

前期研究发现多酚氧化酶(PPO)能正向调控丹酚酸B合成,该研究运用RACE技术,从丹参毛状根中克隆到多酚氧化酶基因(SmPPO,GenBank登录号为KF712274)全长序列,其cDNA全长1 930bp,开放阅读框为1 770bp,编码589个氨基酸。将SmPPO与管状花目其它4个物种进行氨基酸序列比对,在N端类囊体转移结构域中发现都存在2个N-豆蔻酰化位点。在丹参毛状根培养液中加入不同诱导因子,利用实时荧光定量PCR检测,发现该基因在酵母提取物处理中表达量显著上调,但在银离子、抗坏血酸和L-半胱氨酸处理中表达受到明显抑制。运用HPLC技术同步检测毛状根中丹酚酸B含量,显示出与基因表达相同的变化趋势。研究表明,丹参中多酚氧化酶基因(SmPPO)对丹酚酸B的合成具有正向调控作用。     

龙眼胚性愈伤组织DRM1基因的克隆及其定位与表达分析

该研究以龙眼胚性愈伤组织的转录组数据为基础,对龙眼胚性愈伤组织DlDRM1基因进行克隆和生物学信息分析,并检测其在体胚发生过程中不同发育阶段、不同浓度的外源激素(2,4-D、IAA、KT)处理下及不同组织部位的表达,以揭示DlDRM1基因在龙眼体胚发生过程中的功能。结果表明:(1)从龙眼转录组unigene序列筛选获得龙眼结构域重排甲基化酶1基因(命名为DlDRM1)全长序列,并利用RT-PCR法从‘红核子’龙眼胚性愈伤组织中克隆获得DlDRM1基因的cDNA全长序列(GenBank登录号为KY990493);DlDRM1基因cDNA全长2 574bp,包括494bp的5′UTR,184bp的3′UTR,可编码包含631个氨基酸的蛋白质。(2)生物信息学分析显示,DlDRM1是一个不稳定的亲水蛋白,不含信号肽,不存在跨膜结构域,其分子式为C_(3104)H_(4839)N_(851)O_(984)S_(28);序列比对和系统进化分析表明,龙眼DlDRM1与脐橙DRM相似度最高(76.85%),二者亲缘关系也最为接近。(3)实时荧光定量PCR分析发现,DlDRM1在龙眼各组织器官中均有表达,且在果肉中表达量最高,其次是花蕾,在叶中的表达量最低;DlDRM1基因在非胚性愈伤组织中表达量最高,而且在非胚性愈伤向胚性愈伤转变过程中DlDRM1基因的表达量呈逐步下降趋势,说明DlDRM1基因与体胚胚性呈负相关关系,在龙眼体胚发生过程中可能发挥着重要的作用;一定浓度的IAA和2,4-D能够促进DlDRM1基因的表达,而KT则抑制DlDRM1的表达。(4)亚细胞定位结果表明,DlDRM1定位于细胞核和细胞膜上。     

小麦TaLEC1基因的克隆及其表达特性分析

为了探讨LEC1基因在小麦(Triticum aestivum L)非生物胁迫应答中的功能,该研究通过RT-PCR结合RACE技术克隆小麦TaLEC1基因,并采用qRT-PCR方法分析了该基因在小麦不同组织以及不同处理下的表达模式,为深入研究小麦LEC1基因在干旱、高温和高盐胁迫下的响应机制奠定基础。结果表明:(1)成功克隆到小麦TaLEC1基因,该基因cDNA序列全长为1 074 bp,其中5′端非编码区23 bp,开放阅读框为741 bp,3′端非编码区310 bp,编码246个氨基酸,具有典型的CBFD_NFYB结构域。(2)实时荧光定量分析显示,TaLEC1在不同组织间表达差异显著,10 d龄幼苗的叶中表达量最高。(3)TaLEC1基因可被植物激素ABA诱导而上调表达,属于ABA依赖型的表达调控通路。(4)PEG模拟干旱胁迫处理后的0.5～1 h,TaLEC1基因呈上调表达;42℃胁迫处理过程中,TaLEC1基因呈稳定上调表达趋势,并在胁迫处理后12 h和48 h时表达急剧上调,分别为对照的52.8倍和34.5倍;NaCl胁迫处理0.5 h时TaLEC1基因迅速上调表达。研究表明,小麦TaLEC1基因参与ABA依赖的胁迫响应,推测可能在小麦耐受高温胁迫和渗透胁迫过程中发挥着重要的脱水保护功能。     

茶树CsNCED2基因的克隆和表达分析

该研究以铁观音茶树品种叶片为材料,通过RT-PCR技术,克隆了茶树脱落酸(ABA)合成途径关键限速酶——9-顺式环氧类胡萝卜素裂解双加氧酶(9-cis-epoxycarotenoid dioxygenase,NCED)基因的全长cDNA序列。该基因cDNA全长1 931bp,包含1 821bp完整开放阅读框,共编码606个氨基酸残基。NCBI同源分析结果表明,与葡萄VvNCED2相似性最高(78%),命名为CsNCED2(NCBI登录号:MF765770)。氨基酸序列分析显示,其具有NCED家族的FLNO2258保守结构域,以及MIAHPKxDP和HDFAITE保守结构域序列;在保守区存在4个Fe2+活性组氨酸结合位点,N-端含有叶绿体转运肽。实时荧光定量PCR分析表明,CsNCED2基因在铁观音叶、茎和花中表达量较高;白茶萎凋和乌龙茶做青均可以诱导CsNCED2基因显著上调表达;除干旱胁迫抑制CsNCED2表达外,ABA和低温胁迫均能够诱导CsNCED2基因显著上调表达。表明CsNCED2基因在茶树ABA合成代谢以及胁迫响应中发挥重要作用。     

龙眼DCL家族基因的启动子分析及时空表达

为了解龙眼DCL基因的功能,该试验对龙眼基因组数据提取的DlDCL1、DlDCL2、DlDCL3和DlDCL4基因序列进行启动子顺式作用元件及其受miRNA调控的分析;并以龙眼胚性愈伤组织为材料,研究了DlDCLs不同基因成员在非生物胁迫和外源激素处理下的表达情况。结果显示:(1)龙眼DCL基因启动子中除了TATA和CAAT外,还具有大量的光反应元件、激素应答元件、胁迫响应元件、组织特异性调控元件及植物生长发育相关的顺式调控元件,提示龙眼DCL基因启动子转录活性可能受到光、激素信号及逆境胁迫因素的诱导。(2)对调控龙眼DCL基因的miRNA进行筛选,结果显示DlDCL1受miR162和miR1024调控,DlDCL4受miR390和miR396调控。(3)实时荧光定量PCR显示,在一定浓度范围内,外源激素GA3、ABA和ETH均能下调DlDCLs基因的表达,而高浓度ETH处理则显著上调DlDCLs的表达。(4)高浓度蔗糖(6%)处理时DlDCL2、DlDCL3和DlDCL4显著上调表达,而低浓度(0.1%)处理时DlDCL1显著上调表达;不同温度处理下,DlDCL1在34℃时显著上升,DlDCL3随着温度的提高相对表达量逐渐减低;而DlDCL2和DlDCL4表达量差异不明显;NaCl胁迫处理下,DlDCLs在1h处理时表达量下调,但在其他不同时间点则上调表达。研究表明,龙眼DCL基因在外源激素及非生物胁迫处理下,并非是简单的一对一响应,而是存在较为复杂的响应机制。     

龙眼组蛋白去乙酰化酶基因DlHDT1的克隆与特性分析

组蛋白去乙酰化是植物表观遗传调控的重要组成部分,对染色体结构修饰和基因表达调控发挥着重要的作用。为深入探究组蛋白去乙酰化酶基因(histone deacetylase 1,HDT1)在龙眼体胚发生过程中的功能,该研究结合龙眼基因组数据,采用RT-PCR方法克隆得到龙眼组蛋白去乙酰化酶基因(DlHDT1),对其进行生物信息学分析及亚细胞定位观察,同时结合转录组数据分析DlHDT1在体胚发生过程中的FPKM值,并利用qRT-PCR技术检测PEG6000和NaCl处理下DlHDT1的表达模式。结果表明:(1)DlHDT1基因CDS序列全长918 bp,编码305个氨基酸,该蛋白为不稳定亲水性蛋白,不含信号肽和跨膜结构,共含43个磷酸化位点,相对分子量为32 585.54 Da,等电点为4.65;进化树分析显示龙眼DlHDT1与漾濞槭亲缘关系最近(78.76%)。(2)亚细胞定位显示,DlHDT1蛋白定位于细胞核中;顺式作用元件分析发现DlHDT1基因含有大量光响应元件和脱落酸、茉莉酸甲酯等激素及逆境胁迫响应元件;转录组数据显示,DlHDT1在龙眼体胚发生不同时期均有表达,在胚性愈伤组织(EC)阶段表达最低,在球形胚(GE)阶段表达最高。(3)qRT-PCR显示,在PEG6000和NaCl处理下DlHDT1基因,呈下调表达趋势,推测DlHDT1可能参与调控龙眼对干旱及盐胁迫的响应,并存在负调控关系。研究认为,DlHDT1为核定位基因,可能参与龙眼体胚形态建成并在龙眼响应非生物逆境胁迫过程中发挥重要作用。     

龙眼UGD6基因克隆及其表达特性分析

该试验采用RT-PCR和RACE技术,对龙眼多糖合成的关键基因尿苷二磷酸-葡萄糖6-脱氢酶基因(DlUGD6)进行分离克隆、生物信息学分析和亚细胞定位研究,并采用qRT-PCR技术,对其在龙眼体细胞胚胎发生、合子胚发育及不同组织器官中的表达模式进行分析。结果表明:(1)DlUGD6基因的cDNA序列全长1 860bp,包含开放阅读框1 443bp,编码480个氨基酸(GenBank登录号KU198438);生物信息学分析显示,DlUGD6属于稳定的酸性亲水蛋白,不含信号肽,具有跨膜结构和3个典型的保守结构域,属于UDP-葡萄糖/GDP-甘露糖脱氢酶家族;进化树分析表明,DlUGD6与柑橘亲缘关系较近。(2)洋葱内表皮GFP荧光定位观察发现,DlUGD6定位于细胞质;qRT-PCR结果显示,DlUGD6在龙眼非胚性愈伤组织中表达量相对较高,且在其他体胚发育阶段也均有稳定表达;在合子胚发育中子叶胚形成后第8天(S3)和第24天(S7)时表达量最高,整体呈"W"型;在不同组织器官中,DlUGD6在花药和茎中的表达量最高,且整体上生殖器官中的表达水平高于营养器官。研究认为,DlUGD6基因可能参与龙眼生长发育各个阶段中细胞壁多糖合成。     

Coccystes undoxylophus

Ohne Zusammenfassung     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

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.     

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.     

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     

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.