向日葵盐胁迫相关基因的cDNA-AFLP差异表达

目的:研究向日葵盐胁迫前后基因表达的变化,分离并鉴定耐盐相关基因。方法:采用c DNA-AFLP技术分析盐胁迫产生的差异表达基因片段。结果:从256对引物组合中筛选到232对有差异表达的引物组合。用其进行选择性扩增,获得差异表达的上调TDFs 845条。经二次PCR扩增及反向Northern blot验证,获得42个阳性TDFs。对其中12个TDFs进行克隆及序列测定,得到10条TDFs核苷酸序列。经Blastx比对及功能分析,10个TDFs均与应答盐胁迫相关,涉及信号转导相关蛋白、胁迫相关功能蛋白、衰老相关蛋白以及与蛋白相互作用有关的蛋白。结论:利用c DNA-AFLP技术鉴定出一批盐胁迫应答基因,为揭示向日葵耐盐分子机制及指导向日葵耐盐分子育种实践奠定基础。     

茶树冷诱导基因的AFLP筛选及其表达分析

以中小叶茶树良种舒茶早和云南大叶种73-11为材料,于4℃低温处理4 d后,采用AFLP技术筛选出冷诱导下茶树差异表达基因并进行分析.结果显示,实验共获得41个差异片段(TDFs),其中舒茶早产生19条差异条带,73-11产生38条差异条带,两品种共有条带16条.所获得的差异片段按功能可划分为4类,即信号传导蛋白、转录因子、基础代谢相关蛋白、抗逆蛋白质,此外还有一些假设蛋白质以及未知蛋白.利用qRT-PCR对Kh1、Kh3和Kh11差异片段进行验证,结果显示这3个片段均被低温诱导,表达量上升.研究表明,茶树在低温胁迫下的逆境反应非常复杂,涉及多种代谢过程中的众多基因,而且大叶种比中小叶种对低温反应更为敏感.     

基于cDNA-AFLP技术分析不同种源和地域太子参基因差异表达

研究不同种源和地域太子参的基因差异表达,分离并鉴定相关差异基因。采用c DNA-AFLP技术分析2个不同种源和地域太子参差异表达基因。筛选6个引物组合进行扩增,从同种源不同地域及同地域不同种源太子参中分别获得44、38条差异表达转录衍生片段(Trivially distributed file system,TDFs),对差异片段进行克隆、序列测定,分别得到27、25个TDFs核苷酸序列。经过BLASTX程序比对,分别有19、16个TDFs有其对应的显著同源序列,其中有7、6个TDFs为已知功能的蛋白,这些蛋白主要参与植物的生长发育、抵御病虫害、提高植物抗非生物胁迫的能力。本研究为揭示太子参药材品质形成的分子机制提供基础资料。     

芍药种子下胚轴休眠相关基因的cDNA-AFLP分析

旨在探讨芍药种子下胚轴的休眠机理,采用cDNA-AFLP技术对层积0 d和根部露白两个时期的芍药种子进行基因差异表达分析。利用256对引物组合进行扩增,筛选获得3 600个差异表达转录衍生片段(TDFs),成功回收到1 200个TDFs,选取500个TDFs对其克隆测序,共得到42个有效序列。经BLAST比对发现,其中30个TDFs与已知功能基因同源,分别参与芍药种子下胚轴休眠过程中的基因表达调控、信号转导、逆境胁迫、物质与能量代谢等过程,9个TDFs与功能未知基因及假想蛋白同源,另有3个TDFs为无同源序列,可能是新的未知基因,这些基因有助于更好地阐明芍药种子下胚轴休眠机理。     

高粱幼苗水分胁迫诱导表达差异cDNA的研究

以高粱为试验材料,用-0．7MPa的PEG-6000高渗溶液对其幼苗进行水分胁迫处理,利用mRNA差异显示技术分离得到53条高粱水分胁迫诱导表达的cDNA片段,其中包括5个完全诱导表达片段,43个上调片段和5个下调表达片段。经过Reverse Northern验证,筛选出13个差异表达的cDNA片段,并进行克隆测序。经GenBank查询,10个片段序列与已知序列有较高的同源性,3个片段同源性非常低,可能为新基因。     

东乡野生稻及其渐渗系应答低磷胁迫的基因差异表达分析

以东乡野生稻耐低磷渐渗系IL171及其双亲(栽培稻‘协青早B’和东乡野生稻)为试材,采用cDNA-AFLP技术分析其幼苗期应答低磷胁迫的差异表达谱特征,并采用实时荧光定量PCR分析验证差异表达基因的表达特性,为探究东乡野生稻耐低磷胁迫的分子机制、发掘耐低磷相关的基因奠定基础。结果显示:(1)基于17对扩增效果较好的cDNA-AFLP引物分析发现,不同胁迫时间的参试材料与其对照组(正常磷水平)相比,具有很多(20~159个)上调或下调表达的差异片段。(2)与‘协青早B’相比,IL171中特异性上调的差异带有36条,下调表达61条,而东乡野生稻中分别有79条和136条;IL171与东乡野生稻共有的上调差异带为13条,下调差异带有15条。(3)回收纯化其中60条特异性差异表达条带,最终克隆测序获得50个差异表达基因片段TDFs;通过Blast比对和功能分析,可将TDFs分为8类,包括能量与代谢、基因表达调控、信号转导和转录因子等。(4)实时荧光定量PCR验证其中6个TDFs发现,各差异片段的荧光定量PCR表达模式与cDNA-AFLP结果一致,表明该实验的cDNAAFLP差异表达结果可靠,东乡野生稻的部分耐低磷相关基因已成功导入到渐渗系中,是发掘利用东乡野生稻耐低磷相关基因、探究其耐低磷分子机制的重要资源。     

阿尔泰蝠蛾(鳞翅目:蝙蝠蛾科)幼虫低温胁迫转录组分析

[目的]本研究旨在通过转录组测序技术分析低温胁迫引起的阿尔泰蝠蛾Hepialus altaicola Wang幼虫基因转录差异及上调表达基因的主要功能类群和参与的主要代谢通路.[方法]采用Illumina HiSeq TM 2500测序平台对阿尔泰蝠蛾幼虫进行转录组测序、组装,利用Blast软件进行数据库比对和基因功能注释,用DEGSeq R软件包分析4℃低温处理与室内适温饲养试虫的差异表达基因,并对上调表达基因进行GO和KEGG代谢途径富集分析.[结果]经序列拼接后共获得100300个unigenes,总长度81600309 bp,平均长度813 bp,N50长度1719 bp.与7大数据库同源比对,共获得34691(34.59％)条unigenes.低温胁迫转录组分析得到11569个差异表达基因(DEGs),7158条基因上调,4411条基因下调.富集到47个GO类群,217个KEGG途径.其中代谢过程、催化、结合活性类群占有重要比例,核糖体、碳代谢、剪接体等途径显著富集.另外,热激蛋白、昆虫表皮蛋白、海藻糖酶、超氧化物歧化酶等非生物胁迫相关基因显著上调表达.[结论]阿尔泰蝠蛾幼虫低温胁迫转录组分析揭示,代谢过程、细胞过程、生物调节、对刺激的反应等生物学过程相关基因和部分非生物胁迫响应基因显著上调表达,提示蝠蛾幼虫可能从抗氧化防御、分子伴侣、体温调节和维持细胞的渗透平衡等多方面应对低温胁迫.     

阿尔泰蝠蛾(鳞翅目:蝙蝠蛾科)幼虫低温胁迫转录组分析

[目的]本研究旨在通过转录组测序技术分析低温胁迫引起的阿尔泰蝠蛾Hepialus altaicola Wang幼虫基因转录差异及上调表达基因的主要功能类群和参与的主要代谢通路.[方法]采用Illumina HiSeq TM 2500测序平台对阿尔泰蝠蛾幼虫进行转录组测序、组装,利用Blast软件进行数据库比对和基因功能注释,用DEGSeq R软件包分析4℃低温处理与室内适温饲养试虫的差异表达基因,并对上调表达基因进行GO和KEGG代谢途径富集分析.[结果]经序列拼接后共获得100300个unigenes,总长度81600309 bp,平均长度813 bp,N50长度1719 bp.与7大数据库同源比对,共获得34691(34.59％)条unigenes.低温胁迫转录组分析得到11569个差异表达基因(DEGs),7158条基因上调,4411条基因下调.富集到47个GO类群,217个KEGG途径.其中代谢过程、催化、结合活性类群占有重要比例,核糖体、碳代谢、剪接体等途径显著富集.另外,热激蛋白、昆虫表皮蛋白、海藻糖酶、超氧化物歧化酶等非生物胁迫相关基因显著上调表达.[结论]阿尔泰蝠蛾幼虫低温胁迫转录组分析揭示,代谢过程、细胞过程、生物调节、对刺激的反应等生物学过程相关基因和部分非生物胁迫响应基因显著上调表达,提示蝠蛾幼虫可能从抗氧化防御、分子伴侣、体温调节和维持细胞的渗透平衡等多方面应对低温胁迫.     

用mRNA差异显示法分离冷胁迫香蕉幼苗叶片内水杨酸诱导表达的基因

用mRNA差异显示法对7℃低温胁迫3 d后的香蕉幼苗叶片进行研究,回收了18条在低温下水杨酸(salicylic acid,SA)诱导的差异带;反向Northern杂交证明,SA在低温胁迫下能诱导7条差异带高表达.对其中最显著表达的两条差异带(G和A)进行克隆和测序,结果显示,G片段序列与大豆在冷胁迫环境下两个高表达基因片段的部分序列有92%同源性;A片段未发现其同源性基因片段.     

低氧对小麦根端分生细胞核仁结构和功能的影响

为了探讨低氧对小麦根端分生细胞核仁结构和功能的影响,本实验以普通小麦为材料,用低氧水处理其根尖,按常规细胞制片、银染、电镜观察、间接免疫荧光染色和半定量PCR分析等手段开展研究.观察发现:(1)低氧水处理后小麦核仁结构发生膨胀、突出、进而凝集、内部出现空泡、细微结构消失、核仁通道结构异常、甚至解体等一系列变异现象.(2)间接免疫荧光染色技术观察看到,低氧水处理后小麦核仁内的核磷蛋白B23向核质甚至胞质扩散.(3)半定量PCR分析显示,低氧处理后rRNA基因的表达量较对照明显降低,而且C23的表达信号几乎检测不到,表明核糖体RNA和核仁蛋白C23基因的表达均显著下调,低氧严重抑制它们的转录.研究证明,低氧除了对小麦根端分生细胞核仁结构有破坏作用外,还严重抑制核仁的功能.     

小麦根端分生组织细胞核中核仁通道结构的电镜观察

用常规电镜和整体银染电镜观察技术对小麦根端分生组织细胞核进行了研究。发展核仁与其周边染色质之间存在通道结构。初步分析认为,染色体NORs中的rDNA是通过该通道进入到核仁的纤维中心的。     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

Centromere autoantigens are associated with the nucleolus

Because of their importance as target antigens in scleroderma and since all other major autoantigens in scleroderma can be localized to the interphase nucleolus, we were interested in a further investigation of the potential relationship between interphase centromeres and the nucleolus. Using human anticentromere autoantibodies (ACA) from patients with the CREST form of scleroderma as probes in indirect immunofluorescence microscopy, we observed nonrandom interphase "clumping" of centromeres in a distribution suggestive of nucleoli. By double-label immunofluorescence comparing the localization of centromeres to nucleolar proteins Ki-67, fibrillarin, or protein B23 (nucleophosmin), interphase centromeres appeared to be localized around and within nucleoli. A number of different ACA sera were tested on HEp-2, HeLa, PtK2, Indian muntjac, 3T3, and NRK cells, all with identical results indicating colocalization between centromeres and nucleoli. Immunoelectron microscopy revealed that interphase centromeres were distributed free in the nucleoplasm, in contact with the nuclear envelope, in contact with and on the periphery of nucleoli, and totally embedded within the confines of the nucleolus itself. Interestingly, actinomycin D treatment dissociated centromeres from localization within the segregated nucleolus. To determine if interphase centromeres were integral components of nucleoli, nucleoli were isolated according to classical methods. By double-label immunofluorescence, immunoelectron microscopy, and Western blotting, it was demonstrated that centromere autoantigens copurified with isolated nucleoli. These studies offer proof that some interphase centromeres can be associated with, and may even be considered part of, the interphase nucleolus. Furthermore, all of the major autoantigens in scleroderma can now be localized to the nucleolus.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.