草鱼肠道cDNA文库构建及部分ESTs分析

本项研究以雌性草鱼肠道为实验材料,采用非均一化的Oligo-dT引物定向克隆技术构建了草鱼肠道组织的cDNA文库,对文库质量的分析结果表明：cDNA文库的库容量至少为2．3×10^5,重组率达95％,平均插入片断长度大于1000bp。挑取cDNA克隆进行5’端测序,总共进行了1571个成功反应,其中1411条ESTs长度大于100bp,初步拼接得到939个单基因簇（Unigene）,其中包括188个重叠群（Contigs）,751个单拷贝EST（Singletons）。使用BLAST软件将这些序列同GenBank等数据库进行比对、查询和注释,结果显示418条序列有相关同源性,其他的521（55．5％）条序列没有明显的同源性（E-valu≤1．00E-10）,但是也同时揭示出在这些ESTs中能够发现新功能基因的相当可能性。本研究结果对草鱼以及其他鲤科鱼类的消化系统功能基因的筛选和发现具有指导意义。此外,草鱼肠道cDNA文库的构建和EST测序工作将为草鱼基因组计划的实施奠定基础。     

马铃薯泛素结合酶基因StUBC17的克隆与功能分析

泛素结合酶(Ubiquitin-conjugating Enzyme E2,UBC)与植物生长发育和抗病反应密切相关。为了解泛素结合酶基因对植物抗晚疫病的贡献,从马铃薯栽培种"合作88"中克隆出一个E2泛素结合酶基因,命名为StUBC17,并对其受晚疫病菌诱导表达特性及功能进行分析。利用病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)技术降低本氏烟(Nicotiana benthamiana)中StUBC17同源基因(NbUBC)的转录水平,再接种晚疫病菌进行抗病性鉴定。进一步在基因沉默植株上瞬时表达马铃薯抗病基因和其相应的无毒基因(R3a+AVR3a、R3b+AVR3b和Rx+CP)及INF1。StUBC17编码区全长447 bp,编码148个氨基酸,其蛋白分子量为16.52 kD,理论等电点为7.72。表达分析结果表明,StUBC17受晚疫病菌诱导表达。抗病鉴定结果显示,与对照植株相比,NbUBC沉默植株的抗病性显著降低。沉默NbUBC不影响过敏反应(Hypersensitive responses,HR)的发生。StUBC17是植物防御晚疫病所需,但该基因的沉默并不影响R3a、R3b、Rx及INF1介导的HR反应。     

处于性腺不同发育阶段斜带石斑鱼垂体的EST表达差异分析

斜带石斑鱼是重要的海产经济鱼类,在其个体发育过程中存在先雌后雄的天然性反转现象。垂体是调节生长和生殖等生理过程的重要内分泌器官。构建了斜带石斑鱼分别处于卵巢发育起始和性反转后期的垂体SMARTcDNA的质粒文库,并通过测序分别筛选到232个和258个表达序列标签(expressed sequence tags,EST)。将所得EST与GenBank数据库中的序列进行比对,结果表明,处于卵巢发育起始和性反转后期斜带石斑鱼垂体EST中,激素所占比例均为最高,分别为40.5%和34.9%。进一步比较分析了这两个性腺发育时期斜带石斑鱼垂体EST中各种激素相对表达丰度,表明生长/催乳激素家族(GH、PRL和SL)和阿黑皮素原(POMC)表达水平下降;促性腺激素α亚基(GTHα)表达水平急剧上升,促滤泡激素β亚基(FSHβ)、促黄体激素β亚基(LHβ)表达水平上升。     

Genotypic Analysis of Russian Isolates of Phytophthora infestans from the Moscow Region, Siberia and Far East

Phytophthora infestans samples were collected during 1997 and 1998 at multiple sites in Russia from Sakhalin Island in the Far East across Siberia (nine sites, 160 isolates) to the Moscow region (four sites, 325 isolates). In addition, 12 isolates that were obtained previously were included. All isolates were analysed for mating type, and sensitivity to metalaxyl. Isolates from within any of the nine sites outside of the Moscow region were monomorphic for mating type and nearly monomorphic for metalaxyl resistance. In contrast, both A1 and A2 isolates were detected in the Moscow region, and these isolates were also polymorphic for metalaxyl resistance. In two sites in Siberia only A2 mating type strains were detected, in the other six sites in Siberia and in Sakahlin Island, only A1 mating types were detected. A subset of isolates ( n =191) was also analysed for pathotype (virulence to 10 R-genes, each in a distinct differential genotype). All isolates were highly complex (mean number of virulences approximately 8.4 of a maximum number of 10). All isolates ( n =43) from Sakahlin Island were virulent to all 10 of the R-genes tested. A further subset of isolates ( n =70, including 12 isolates collected before 1997) was analysed for genotype at the Glucose- 6-phosphate isomerase and Peptidase loci, mtDNA haplotypes, and RFLP pattern using the RG57 probe. The US-1 clonal lineage (previously dominant) was not detected in the 1997–98 sample. The populations of P. infestans near Moscow in 1997 and 1998 was highly diverse with 15 unique genotypes (including both mating types) among a sample of 18 isolates. In contrast, the populations of P. infestans in Siberia had limited diversity, with only three multilocus genotypes detected and most populations were dominated by the SIB-1 clonal lineage. This lineage accounted for 31 of the 39 strains collected in Siberia that were assayed for multilocus genotype.     

Expressed Sequence Tag Analysis of Vanadocytes in a Vanadium-Rich Ascidian, <Emphasis Type="Italic">Ascidia sydneiensis samea</Emphasis>

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10⁷ times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.     

The Relative Importance of Seed-borne Inoculum to Common Scab Disease of Potato and the Efficacy of Seed Tuber and Soil Treatments for Disease Control

The importance of both seed and soil-borne inoculum in the epidemiology of common scab disease under Australian conditions was clearly demonstrated. In field trials the severity of disease in harvested potatoes was directly related to the severity of disease on the planted seed tubers. Chemical seed dressing treatments were assessed for common scab disease control under field conditions in four trials over 5 years. Where seed treatments were applied to both diseased and visibly clean seed pieces significantly more disease was found in tubers harvested from diseased seed than the corresponding clean seed treatment. In all but one trial, the treatments applied to diseased seed significantly reduced the incidence of common scab. Fluazinam, flusulfamide (at elevated rates), fenpiclonil, pentachloronitrobezene and mancozeb seed treatments were particularly effective as seed dressing treatments. Applications to visibly clean seed failed to significantly diminish disease levels below that found on untreated seed. Preliminary investigations of some chemical soil treatments gave disappointing levels of control.     

高海拔生境下怀玉山高山马铃薯和本土农家薯的全基因组重测序分析

为了探究怀玉山高山马铃薯只限于高海拔生境下的适应机制,本研究以高海拔生境下怀玉山高山马铃薯和怀玉山本土农家薯采后块茎萌发的芽为材料,进行了全基因组重测序分析。结果表明:HAP的总SNP数量为2 835 211,SNP的密度为3.925 212,编码区nsSNP总数为104 456;LAP的总SNP数量为3 968 618,SNP的密度为5.427 855,编码区nsSNP总数为141 060。经CV分析后,LAP和HAP共有319个nsSNP关联了315个基因,其中76个基因具有超过1个的nsSNP。nsSNP的防御反应、蛋白氨基酸磷酸化、蛋白激酶等GOterms显著富集。HAP的总Indel数量为159 797,编码区的Indel数量为1 850;LAP的总Indel数量为154 291,编码区的Indel数量为1 714;两组的总Indel数量远远小于两组的总SNP数。经差异分析后,共有1 629个Indel关联到了726个基因。Indel的防御反应、胁迫应答、细胞凋亡等GOterms显著富集。nsSNP和Indel的GOterms大多与高海拔生境和光合作用相关。nsSNP和Indel分布在端粒附近相对于中心粒具有更高密度的变异。LAP和HAP共获得了1 490个CNV,其中缺失(Deletion)为610个,中性(Neutral)为548个,扩增(Amplification)为332个。本研究结果可为高海拔生境下怀玉山高山马铃薯的SNP和Indel相关标记的开发、优异基因的挖掘提供重要理论依据。     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.     

一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

羊种布鲁氏菌侵染HPT-8细胞cDNA文库的构建及EST序列分析

拟建立羊种布鲁氏菌侵染HPT-8细胞的cDNA文库。分别提取布鲁氏菌侵染20min、1h、2h、3h、4h后HPT-8细胞总RNA,逆转录合成cDNA,同源重组法构建布鲁氏菌侵染HPT-8细胞的cDNA文库,测定其库容量和重组率,对文库63个克隆进行测序分析,采用BLASTx和BLASTn进行序列同源性比对,并将63个基因进行了功能分类。结果得到的羊种布鲁氏菌侵染人胚胎滋养层细胞HPT-8cDNA文库的库容为1.43×106,重组率是96.92%,插入片段大小为0.2-5.0kb。在63个基因中,与转录、能量代谢、运输及细胞因子相关的基因所占比例较高。构建的羊种布鲁氏菌侵染HPT-8细胞的cDNA文库,为研究宿主细胞受体和布鲁氏菌入侵途径,进一步了解布鲁氏菌的致病机理奠定了基础。     

马铃薯卷叶病毒基因间隔区的克隆及序列分析

根据已报道的马铃薯卷叶病毒基因组序列．设计合成一对特异性引物,以马铃薯卷叶病毒中国分离株（ＰＬＲＶ－Ｃｈ）的ＲＮＡ为模板,反转录合成ｃＤＮＡ第一条链,经ＰＣＲ扩增后克隆于ｐＵＣ１９质粒中,进一步用ＰＣＲ鉴定、限制酶切分析和序列分析,结果表明：ＰＬＲＶ－Ｃｈ基因间隔区由１９７个核苷酸组成,与国外报道的荷兰ＰＬＲＶ－Ｎ加拿大ＰＬＲＶ－Ｃ,澳大利亚ＰＬＲＶ－Ａ,苏格兰ＰＬＲＶ－Ｓ各株系核苷酸序列具有很高的同源性,同源率依次为９９％、９８％、９３％、９８％。     

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。