抑制差减杂交分离赤霉病菌诱导的小麦特异表达基因

为了全面探索小麦赤霉病抗性机理,以抗赤霉病小麦品种‘苏麦3号’及其感病的近等基因系(Hwo4)为实验材料,构建了一个‘苏麦3号’受赤霉病菌诱导表达的正向差减杂交文库。随机选取了141个阳性克隆测序,共获得133条通读EST。将获得的EST去除载体及接头序列后,利用CAP3软件进行聚类分析,133条EST被聚成90个序列重叠群(contigs),片段大小介于106~643 bp间,平均长度为274 bp。利用NCBI的Blastx软件对序列进行蛋白序列同源性比对,结果显示76条序列在蛋白数据库中可以找到同源序列,功能涉及能量代谢、物质代谢、疾病/防御、转录及细胞结构等。其中以能量及物质代谢相关的基因数量最多,分别占总EST的21.1%及17.1%;其次是与抗病及防御反应有关的EST,数量占总EST的15.8%。进一步的分析表明与抗病及防御相关的EST功能主要涉及抗氧化、细胞解毒及相关物质的代谢。     

利用基因芯片数据挖掘宫颈癌相关基因的EST片段

目的:利用基因芯片数据,探讨宫颈癌在分子水平上的发病机制,挖掘肿瘤相关基因EST片段,探索恶性肿瘤标志物,为肿瘤防治找到新的有效手段。方法:从基因芯片数据库GEO(gene expression omnibus)中获得GSM99077基因芯片数据,利用该数据筛选出宫颈癌相关基因的EST片段;然后通过NCBI中的在线BLAST软件找到与之相匹配的同源序列,对这些同源序列进行生物学功能分析,找到与肿瘤的相关性。结果:共发现宫颈癌组织与正常宫颈组织差异表达EST共127条,其中上调的106条,下调的11条,这些差异表达EST的同源序列的转录产物参与转录、翻译、细胞增殖分裂及细胞信号传导等过程。结论:基因芯片能有效、高通量地获取生物内在信息,通过对基因芯片数据再挖掘,可发现宫颈癌的发生涉及多个基因共同作用。     

家蚕母性基因的表达序列标签分析

为了研究家蚕Bombyx mori卵中储存的母性遗传信息,以未受精的肾脏型和正常型蚕卵为材料构建了cDNA文库,并对其随机测序,共获得391条表达序列标签(EST)序列,经拼接后得到374条非重复序列。生物信息学分析发现,172个非重复序列与国际数据库GenBank和silkbase中的已知基因具有较高的相似性。除3个已知母性基因外,其余主要与DNA复制、能量代谢、信号转导、转录、蛋白质结构以及胚胎发育中个体形成等相关。该结果可为进一步研究家蚕胚胎早期发育的调控提供基础数据。     

白桦雌花序抑制性消减文库构建及EST分析

为研究白桦雌花序发育过程中特异基因的表达,以白桦雌花序样品为tester,雄花序样品为driver,利用SMART策略构建了白桦雌花序抑制性消减（SSH）文库。构建SSH文库的重组率为72%,插入片段的平均长度为400 bp左右。随机挑选文库克隆测序,获得150条EST序列,这些序列被GenBank的dbEST数据库收录,收录号为EE284580-EE284681,EE595316-EE595363。通过BlastX对EST进行功能注释,并对其中同源性较高的111条EST按功能进行分类,EST功能涉及了代谢、细胞防御、转录调节、能量代谢及信号传导等途径。发现了多个已知的控制花发育相关的EST,它们占已知功能EST的21%其功能涉及到调控花序形成和花分化、调控花粉与柱头亲和性以及调控花粉管发育等,包括MADS-box、S-locus F-box等基因。这些EST的获得为了解白桦花期基因表达,白桦花发育相关基因克隆和功能解析奠定了基础。     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号（Stylosanthes guianensis‘Reyan2’）对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导基因,利用抑制消减杂交（SSH）技术构建在300μmol·L-1铝胁迫下正向cDNA文库。挑选插入片段大于300bp的600个克隆进行测序,共获得504条表达序列标签（EST）。序列重复性分析表明,其中12.1%的EST只有1次重复,61.4%的EST有2-16次重复,重复出现次数较高的EST是细胞色素P450（53次,占10.5%）、病原诱导型胰蛋白酶抑制剂（44次,占8.7%）和衰老相关蛋白（37次,占7.3%）。BLASTX分析显示,504条EST中有97种非冗余基因,其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因,16个是新发现的铝胁迫相关基因。SSHcDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

''陕麦139''受条锈菌抑制表达相关基因的分析

以‘条中32’接种前后的小麦抗条锈病种质‘陕麦139’幼苗叶片为材料,利用抑制消减杂交技术,构建了条锈菌接种48 h小麦抗病品种叶片的抑制表达SSH-cDNA文库,通过测序以了解条锈菌侵染后被抑制表达的相关基因。从构建的文库中随机选取48个阳性克隆,进行测序,经过序列拼接等,获得高质量EST序列34条(Gen-Bank序列号为EL930132-EL930165),序列比对分析表明其中31条EST序列与已知功能的基因序列同源性较高,主要涉及植物的能量代谢途径、膜转运、信号传导及核酸加工等方面的基因,包括光合系统Ⅱ抗性蛋白D1、碳酸酵素、线粒体磷酸盐转运子、苹果酸脱氢酶、过敏性诱导反应蛋白和rRNA核酸内切酶等相关基因。此外,获得未知功能的EST序列3条。研究表明,小麦在条锈病菌侵染初期,许多基因的正常表达调控受到影响,如能量代谢和寄主细胞结构木质化,阻止小麦抗病过敏性反应等。     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号(Stylosanthes guianensis ‘Reyan 2’)对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导 基因, 利用抑制消减杂交(SSH)技术构建在300 μmol·L^–1铝胁迫下正向cDNA文库。挑选插入片段大于300 bp的600个克隆进行测序, 共获得504条表达序列标签(EST)。序列重复性分析表明, 其中12.1%的EST只有1次重复, 61.4%的EST有2–16次重复, 重复出现次数较高的EST是细胞色素P450(53次, 占10.5%)、病原诱导型胰蛋白酶抑制剂(44次, 占8.7%)和衰老相关蛋白(37次, 占7.3%)。BLASTX分析显示, 504条EST中有97种非冗余基因, 其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因, 16个是新发现的铝胁迫相关基因。SSH cDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

基于冗余的序列检测东北虎表达序列标签数据多态性

以本研究室103测序获得的EST序列(expressed sequence tags)和公共数据库(GenBank)中下载的共29 832条东北虎EST序列为分析内容,利用生物软件DNA star对序列进行拼接,得到3 301个重叠群(contigs),含有4条EST以上的187条contigs (拼接序列)中,共获得489个候选SNP位点,其中颠换型、转换型和单碱基的插入与缺失分别为269个,182个和148个。SNP的平均出现频率为5.2 SNP/contig。采用Quality SNP软件,在22个EST重叠群中检测到90个可信度高的与生长发育相关的SNP。其中包括转换型31个,颠换型26个和缺失型33个。对含有SNP的基因进行了GO注释。共获得了689个注释。KEGG通路分析表明共有357条contigs被注释到了Enzyme commission (EC)号,这些基因参与了68条代谢通路。对所鉴定的SNP进行注释表明,本研究所开发的SNP将促进东北虎生长性状的分子基础研究。     

白叶枯病菌胁迫下云南普通野生稻SSH文库的构建及抗病相关基因分析

以云南普通野生稻为材料,利用抑制差减杂交技术（SSH）,构建了白叶枯病菌胁迫的云南普通野生稻特异表达基因的差减文库.通过对文库所有阳性单克隆进行测序,聚类分析后共获得494条高质量的表达序列标签（EST）.经过BlastN分析,有417条与已知功能的序列有较高同源性;经BlastX分析,有104条EST与未知功能蛋白或假定蛋白有较高相似性,49条EST未能找到同源匹配,341条EST与已知功能蛋白有较高同源性.初步分析发现,这些基因主要涉及能量代谢、蛋白质代谢、核酸代谢、防御与抗逆应答反应、信号转导、光合作用及膜运输等代谢过程.使用半定量RT-PCR研究了7个可能与白叶枯病抗性相关的EST序列在云南普通野生稻对照和白叶枯病菌处理的叶片中的表达情况,并获得这些基因的表达谱.结果发现,克隆编号为OR7,OR68和OR826的EST受白叶枯病菌胁迫诱导上调表达,其中OR826 EST在蛋白数据库中无同源序列,可能是一类新的白叶枯病抗性基因,而组成型表达的OR143 EST在对照和接菌处理的叶片中均能检测到其mRNA的表达,但其表达量在白叶枯病菌胁迫48 h后逐渐增强,推测这些基因直接参与了云南普通野生稻抗病防御反应.本研究为从云南普通野生稻中发掘和克隆新的白叶枯病抗性基因提供了理论依据,为进一步研究云南普通野生稻抗白叶枯病的分子机制奠定了基础.     

基于葡萄EST数据库糖代谢途径有关基因的分析

以拟南芥糖代谢相关酶基因序列作为探针,对葡萄EST数据库进行同源检索筛选,获得相应的同源EST序列,并以葡萄果实cDNA为模板,通过PCR反应,对葡萄糖代谢途径相关基因进行了分析。进一步预测了葡萄糖代谢途径相关酶编码基因个数,分别位于1、4、5、6、7、8、11、12、13、16、17、18、19号染色体,另有3条序列未能定位到染色体,并对编码基因表达强弱进行了分析,为深入了解葡萄果实内糖代谢的机理提供一定的工作基础,也可以为如何调控果实糖代谢以及提高葡萄果实品质提供理论依据。     

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.     

Comparative analysis of expressed sequence tags from Sesamum indicum and Arabidopsis thaliana developing seeds

Sesame (Sesamum indicum) is an important oilseed crop which produces seeds with 50% oil that have a distinct flavor and contains antioxidant lignans. Because sesame lignans are known to have antioxidant and health-protecting properties, metabolic pathways for lignans have been of interest in developing sesame seeds. As an initial approach to identify genes involved in accumulation of storage products and in the biosynthesis of antioxidant lignans, 3328 expressed sequence tags (ESTs) were obtained from a cDNA library of immature seeds 5-25 days old. ESTs were clustered and analyzed by the BLASTX or FASTAX program against the GenBank NR and Arabidopsis proteome databases. To compare gene expression profiles during development of green and non-green seeds, a comparative analysis was carried out between developing sesame and Arabidopsis seed ESTs. Analyses of these two seed EST sets have helped to identify similar and different gene expression profiles during seed development, and to identify a large number of sesame seed-specific genes. In particular, we have identified EST candidates for genes possibly involved in biosynthesis of sesame lignans, sesamin and sesamolin, and also suggest a possible metabolic pathway for the generation of cofactors required for synthesis of storage lipid in non-green oilseeds. Seed-specific expression of several candidate genes has been confirmed by northern blot analysis.     

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.