没顶淹涝早期水稻基因差异表达分析

水稻（Oryza sativa L）耐淹品种FR13A在分蘖期进行没顶淹涝处理。分别取淹涝0h,2h,4h,8h,16h,32h的叶片进行mRNA差异显示分析,获得8个差异表达的cDNA片段。利用反式Northern斑点杂交法去除4个假阳性片段,对4个阳性片段（DF8、DF9、DF10、DF11）测序后进行同源性分析,其中一个片段（DF8）的蛋白产物与WD-40重复蛋白高度同源,另一个片段（DF11）的蛋白产物与植物生长素应答蛋白有较高同源性,其余2个没有找到同源蛋白质序列。     

显性核不育亚麻可育、不育花蕾mRNA差异表达研究及差异片段分析

利用mRNA差异显示技术,以显性雄性核不育亚麻不育系分离出的可育株和不育株为材料,对可育、不育花蕾中基因差异性表达进行了研究.对回收的差异片段进行反向Northern杂交验证,获得了7个阳性差异表达基因片段,并对它们进行测序和BLAST分析,最后确定了3个候选基因片段G-E07-100、G-E07-830和G-E07-330.序列分析结果表明:G-E07-100与蓖麻(Ricinus communis)的GTP-结合蛋白基因部分序列高度(90%)同源;G-E07-830与梨(Pyrus pyrifolia)的β-木糖苷酶基因高度同源(80%),并且还包含1个糖基水解酶家族C-末端的保守结构域;G-E07-330与亚麻(Linum usitatissimum)LuP12025D10 cDNA高度(89%)同源.进一步的Northern杂交结果表明3个候选基因均在不育花蕾中表达.     

小麦苗期水分胁迫诱导差异表达cDNA的研究

以小麦幼苗为材料 ,采用mRNA差异显示方法和银染技术 ,对经过用 16 % (- 0 .5MPa)PEG - 6 0 0 0溶液处理不同时间而诱导表达的小麦基因进行分离 ,共得到cDNA差异片段 5 2条。经ReverseNorthern验证 ,检出阳性表达片段 15个 ,克隆并测序。经GenBank查询 ,11个片段序列与已知序列有较高的同源性 ,4个片段同源性非常低 ,可能为新基因。     

辣椒冷诱导差异表达基因分析

采用cDNA-AFLP技术分析了4℃低温诱导前后耐寒辣椒(Capsicum annuum L.)品系P70的基因表达差异谱.结果获得了120个差异片段,对阳性克隆中的12个差异片段进行测序和Blast-x比对,发现其中有4个片段(KH-1、KH-2、KH-3和KH-4)与抗逆性相关,其碱基数分别为185、160、269和511 bp,4个片段分别与编码抗坏血酸过氧化物酶基因的同源性达98%、与细胞色素p450基因的同源性达98%、与牛血清蛋白(BSA)基因的同源性达94%、与水孔通道蛋白(AQP)基因的同源性达90%.4个差异基因表达模式为:基因KH-1、KH-2、KH-3上调表达,KH-4下调表达.     

小麦拔节过程中基部茎节的基因差异表达

采用差异显示逆转录PCR(DD-RT-PCR)技术分析小麦基部茎节伸长过程中基因表达差异的结果表明,小麦拔节前和拔节后基部茎节中存在明显的基因表达差异(包括质和量的差异).从具有质的差异表达基因中挑选了4个基因进行cDNA片段(编号为WSRI～4)的克隆,测序和GenBank数据库序列同源性比对分析,结果显示,WSRI与大麦中编码Ser/Thr蛋白激酶的序列具有很高的同源性,WSR2的功能未知,WSR3高度同源于玉米中码Ty3/gypsy类Kg-座子反转录酶的序列,WSR4高度同源于小麦的端粒关联DNA.半定量RT-PCR技术分析拔节过程中克隆基因WSRl～4表达特性显示,除了WSR4以外,其余3个基因的表达模式和通过DD-RT-PCR分析所展示的差异表达趋势基本一致.     

差异显示法分离水稻抗稻瘟病相关基因

采用mRNA差异显示技术,分析水稻稻瘟病抗源材料“地谷”叶片受稻瘟病菌侵染前后的基因的表达差异,获得87个差异片段。对这87个差异片段进行了回收、重扩增与克隆,并对其中的81个片段进行了杂交鉴定。斑点杂交结果证实其中6个片段受稻瘟病菌诱导表达。进一步克隆测序并进行数据库比对分析表明其中一个与水稻4号染色体中一推测的苹果酸合成酶高度同源,一个与水稻11号染色体上的RPR1基因高度同源,RPR1基因具有保守的NBS-LRR结构,并与水稻防卫反应的信号传导有关;另一个与水稻第6号染色体上一推测的硫氧还蛋白高度同源,其余3个为新的cDNA片段。     

牡丹CTR1基因家族基因片段及其cDNA 3′-末端的克隆与序列分析

以牡丹品种‘洛阳红’(Paeonia suffruticosa‘Luo Yang Hong’)花瓣为材料,用CTAB法提取总RNA,根据已报道的CTR1(Constitutive Triple Response 1)蛋白的保守氨基酸序列设计简并引物,通过RT-PCR扩增得到3条牡丹CTR1基因同源片段。利用其已知中间序列,通过3′快速扩增cDNA末端技术(3′RACE)及序列拼接,最终得到了这3个基因片段除5′末端外的全部cDNA序列,分别命名为PsCTR1、PsCTR2和PsCTR3。分析结果表明,由PsCTR1、PsCTR2和PsCTR3基因翻译的氨基酸片段与拟南芥和番茄的CTR1蛋白氨基酸序列的同源性均较高,分别为88%和85%;61%和61%以及70%和69%。     

小麦光温敏核雄性不育相关基因的G-box家族引物差式分析

用G-box家族引物对小麦光温敏核雄性不育系农大3338在可育与不育光温条件下进行mRNA差异显示,结果表明,在育性转换时期,这两种条件下的基因表达存在显著差异。回收了12个质的差异片段并进行反Northern印迹杂交验证,然后对5个阳性克隆片段HT1-G10、HT1-G3、HT2-G2、HT1-G4和HT2-G5进行了测序,同源比较显示：HT1-G10与小麦（Triticum aestivum）叶绿体基因rbcL和atpB的部分序列高度同源（96％）;HT1-G3与小麦（Triticum aestivum）组蛋白H2A基因高度同源（88％）;另3个片段为新基因片段。对这些基因片段的分析为揭示光温敏核雄性不育的发育机理提供了一些有效证据。     

禾谷镰孢菌α-微管蛋白基因克隆及其与多菌灵抗药性关系分析

根据禾谷镰孢菌参考菌株NRRL310 84 (PH 1)的α- 微管蛋白基因核苷酸序列设计 4对引物 ,采用PCR方法克隆并测序了禾谷镰孢菌 (Fusariumgraminearum)对多菌灵 (MBC)不同敏感性表型的 6个中国菌株的α 微管蛋白基因全序列。DNA序列对照表明中国的 3个敏感菌株和 3个抗药菌株的α- 微管蛋白基因核苷酸序列同源性没有差异 ,多菌灵抗药性与α- 微管蛋白无关。该基因全长 1718bp ,含有 6个内元 ,编码 4 4 9aa ;与NRRL310 84的α- 微管蛋白基因核苷酸序列同源性为 99% ,存在 5个差异核苷酸 ,与其所编码的氨基酸序列同源性为 99 78% ;与其他 6种真菌α- 微管蛋白基因所编码的氨基酸序列同源性为 37%～ 86 %。     

二化螟β1微管蛋白基因cDNA序列的克隆与序列分析

微管是真核生物体内分布最为广泛的一类蛋白,是由a-和β-两种不同的微管蛋白组成的异源二聚体.微管参与许多细胞功能,如细胞形态发生、细胞生长和分裂等.以二化螟3龄幼虫为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到该虫的β微管蛋白基因的cDNA序列一条.cDNA序列含1 862个碱基,开放读码框1 344个碱基,编码氨基酸447个,分子量约为50.2kD,等电点4.82.氨基酸序列中1～4个氨基酸MREI为β微管蛋白转录后调控信号,是微管蛋白特异片段;氨基酸序列的140～146GGGTGSG位存在一个微管蛋白标志信号片段.序列比对表明,克隆的β微管蛋白基因与其他昆虫的β微管蛋白基因在核苷酸和氨基酸水平上都是高度同源的,与家蚕Bombyx moriβ1微管蛋白的氨基酸序列同源性达到99.1%,与烟草天蛾β1微管蛋白的氨基酸序列同源性达到98.7%,与果蝇Drosophila melanogasterβ1微管蛋白的氨基酸序列同源性达到96.9%.该基因cDNA序列已经登录Genbank并获得登录号为EU429675.     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.     

cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell     

加速衰老小鼠脑组织中的衰老相关基因的表达

从分子水平上研究衰老对大脑的影响有助于揭示机体衰老的分子机理 ,也有助于揭示衰老相关性脑功能异常的发生过程。本研究应用DDRT PCR方法研究衰老相关基因在SAM (Senescence acceleratedmouse)小鼠脑组织中表达的变化情况。在SAMR1TA、SAMP8/Ta、SAMP1 0 /Ta三个品系中 ,通过比较不同鼠龄SAMP1 0 /Ta (2、 4、 1 2、 1 8月龄 )的基因表达情况 ,发现在 4月龄和 1 2月龄分别有一个差异表达片段 ;对不同鼠龄的SAMP8/Ta (2、 4、 1 1月龄 )经差显比较 ,发现在 2月龄和 1 1月龄各有一差异表达片段。在不同品系的比较中发现了 1 6个差异性片段 ,分别属于SAMP1 0 /Ta (3个 )、SAMP8/Ta (6个 )和SAMR1TA (7个 )。测序结果经检索显示 ,它们分别与下列基因转录产物同源 :热休克识别蛋白 70、ATP依赖性线粒体RNA螺旋酶、DleumRNA、小鼠X染色体RP2 3 334C4克隆DNA序列、还原型辅酶Q 细胞色素c还原酶复合物 7 2kD亚单位、 6 0S核糖体蛋白L2 1、FIS、苯基烷基胺钙离子拮抗物结合蛋白、岩藻糖基转移酶 9、胶质细胞源性神经营养因子家族受体α1、内切核酸酶 /逆转录酶、PER1蛋白相关超级融原核蛋白、中心体蛋白CG NAP、转铁蛋白重链基因、巢蛋白 2基因、DNA依赖性蛋白激酶催化亚单位基因 prkdc     

Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon.

The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli. We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions. Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E. coli genetic map. Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon. The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide. The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon. The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein. The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999