人TRAIL基因胞外段的化学合成和克隆

根据大肠杆菌高表达序列重新设计了人肿瘤坏死因子相关的凋亡诱导配体(TRAIL)胞外段的基因编码区的核苷酸序列, 应用化学合成的方法合成了多个互补DNA片段,经互补片段分别退火和连接后, 将完整编码区克隆于pMD18-T质粒中,进行DNA 序列分析,获得了与设计序列完全一致的含有完整编码区的人TRAIL胞外段基因,并成功构建了高效的TRAIL胞膜外段原核表达载体pTWIN1/TRAIL111-281.     

黑子南瓜中STK类抗病基因同源序列的克隆及序列分析

 黑子南瓜是一种具有较强抗病性的葫芦科植物,依据所克隆的植物丝氨酸/苏氨酸蛋白激酶类抗病基因中的保守氨基酸序列合成相应的简并引物,从黑子南瓜基因组DNA中通过PCR克隆到了一些STK类的R-片段,经相关软件分析发现其中之一可以编码完整的氨基酸序列,可能来源于黑子南瓜中某个抗病或抗病相关基因.该基因片段与来源于其他已知基因的相应序列比较发现同源性都比较低,属于一个新的STK类R-片段家族成员.从黑子南瓜中克隆STK类片段,将为进一步从该植物材料中获得STK类抗病基因提供新线索.     

人转铁蛋白基因的克隆及序列分析

目的:克隆人转铁蛋白基因并对其编码序列进行分析.方法:以人胎肝cDNA为模板,利用PCR方法克隆人转铁蛋白基因;通过与基因组序列对比分析基因组结构;通过TargetP 1.1和SignalP 3.0预测信号肽;通过Clustal X(1.81)进行蛋白序列联配.结果:PCR扩增了一个长2 160 bp的基因片断,序列分析表明其覆盖了完整编码框,编码由698个氨基酸组成的人转铁蛋白.进一步分析发现人转铁蛋白基因有19个外显子和18个内含子,编码人转铁蛋白N端具有19个氨基酸组成的信号肽序列.人转铁蛋白与猩猩、猴子、兔子和老鼠的转铁蛋白氨基酸相似率分别为94%、91%、78%、73%.生物信息分析表明,人转铁蛋白含有高度保守的参与蛋白二硫键形成的半胱氨酸以及铁离子结合位点,有两个序列较同源的结构域.结论:成功克隆人转铁蛋白基因,人转铁蛋白与其它物种转铁蛋白同源.     

癌症相关基因选择性剪接进化数据库的构建

选择性剪接是真核生物基因调控的基本调节机制之一,与各种类型的生理和病理活动相关.癌症相关基因的不正常剪接可能与多种癌症的发生发展有关.作为选择性剪接进化的主体,选择性剪接外显子展示了其在不同物种中多样的进化功能.本文系统整理了2 989个癌症相关基因的各项功能,通过比较基因组学的分析方法总结了癌症相关基因的选择性剪接外显子的功能和进化关系,建立了癌症相关基因选择性剪接进化数据库(ASeeDB数据库).ASeeDB包含癌症相关基因外显子区域的进化保守性、结构域预测、Ka/Ks值、以及基因、转录本、外显子区域3个层次的表达量统计等信息,结合这些信息用户可以方便的检索有研究意义的基因或者外显子.外显子区域蛋白质结构域的预测可以帮助了解其可能的功能,而外显子是否存在选择性剪接又可以推及包含该外显子的转录本是否具有相似的功能以及推测剪接是否会对蛋白质功能发生影响.数据库提供的物种间的序列比对可以帮助用户发现没有注释的外显子区域或者是保留的失去功能的外显子.相比于基因层面的Ka/Ks,数据库提供的外显子层面的Ka/Ks对于发现适应性进化事件具有更高的敏感性,可以更加方便地预示基因中未知功能的区域.     

棉花半胱氨酸蛋白酶抑制剂基因的克隆、表达与活性研究

通过从已构建好的突变体湘棉-18的cDNA文库中筛选分离出一个半胱氨酸蛋白酶抑制剂基因的全长cDNA序列,经测序和序列分析,发现该片段包含一个完整的开放阅读框,长378个碱基,编码125个氨基酸,氨基酸序列中包含了一个半胱氨酸蛋白酶抑制剂家族的保守区段Gln-Val-Val-Ala-Gly;信号肽预测发现该序列包含一个N端信号肽,保守区分析该基因编码的氨基酸包含一个半胱氨酸蛋白酶抑制剂相似区域.半胱氨酸蛋白酶抑制剂在大肠杆菌中表达重组蛋白,在条件为30℃,IPTG 5mmol/L,时间24～36h时,CPI实现最优表达,Western Blotting分析表明表达的蛋白正确.通过木瓜蛋白酶活性抑制实验,结果表明该重组CPI具有一定的酶活抑制作用.     

2001-2010年东洞庭湖湿地NDVI指数的水位响应

利用TIMESAT拟合2001年至2010年东洞庭湖的MODIS-NDVI数据,获得整个东洞庭湖湿地A及湿地西侧B和南侧C三个区域的NDVI时间序列.通过分析NDVI时间序列与水位变化关系、绘制样本自相关曲线及计算Spearman秩相关系数(ρs),研究了东洞庭湖湿地植被覆盖和水位变化两方面功能状态,并探讨东洞庭湖湿地植被覆盖对水位变化的响应.研究表明:1)东洞庭的植被覆盖状况受水位作用是以年为周期,每周期内的4月及10月底出现一个峰值,丰水期7月份出现一个降低过程;二次多项式拟合显示植被覆盖峰值的水位临界点在24~25m左右.2)三个研究区植被受湖泊水位作用大小关系是:A区B区C区,其主要影响因素是植被类型与区域高程的差异.3)在2003,2006及2009年这三个三峡的重要蓄水年,位于水陆交错带的NDVI值与水位的秩相关系数变化较大;三峡蓄水水位从135m上升至175m的这三年,三区域全年的ρs分别增长至0.641,0.687,0.518,相关性增强.三峡蓄水对东洞庭湖植被覆盖与湖泊水位关系产生一定影响.     

具有σ-局部可数cs* - 网的空间

利用ponomarrv's 方法及msss 映射的定义, 研究了广义度量空间中的点可数覆盖问题. 证明了具有σ-局部可数cs^* - 网的空间可以刻划成度量空间的序列覆盖映射和强序列覆盖映射下的象, 从而使关于对σ- 局部可数族的讨论趋于完整.     

水稻白叶枯病菌hrp调节基因hrpG的鉴定及其变异分析

根据水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)hrp基因的序列设计引物,通过PCR方法从水稻白叶枯病菌JXOⅢ和PXO99A菌株中扩增得到核苷酸序列完全一致的hrpG基因.以该基因为探针与JXOⅢ/pUFR034基因组文库中含有hrpX克隆pUHRX245(36.8 kb)的4个亚克隆进行southern blot分子杂交,确定在亚克隆pB1中1.3 kb大小的片段和AE4(3.0 kb)片段中存在hrpG同源序列.测序结果表明,在pB1中有586个碱基序列,在AE4中有206个碱基,共同构成完整的hrpG基因,且与已知的hrpX基因是毗邻的.以同样方法从水稻白叶枯病菌PXO99A菌株的hrp-突变体M16中扩增得到hrpG基因对应位置的同源突变序列.序列分析表明其有意义突变为541位的C→T的突变,从而导致亮氨酸变为苯丙氨酸(Leu→Phe).将hrpG基因和突变序列分别导入突变体M16中,转移结合子M16/hrpG(PXO99A)恢复了其在烟草上的过敏反应和在水稻上的致病性,而突变序列的结合子M16/hrpG(M16)则不能使其恢复功能,从而确定M16是由于单个碱基的突变所引起的功能突变.经过对基因及其产物的比对发现,对于第Ⅱ组hrp调节基因hrpG的变异,种间变化明显高于种内不同致病变种的变化.     

牦牛SRY和TRO基因部分序列克隆与测序分析

对牦牛SRY和TRO的部分基因克隆和序列分析,以期为牦牛X精子和Y精子鉴定、性染色体的基因定位以及分子标记辅助选择研究提供理论依据.从牦牛和西门塔尔牛冷冻精液中提取DNA,用特定引物对SRY、TRO基因部分序列进行扩增并进行TA克隆和测序.结果表明,这两个基因区域在牛种中有极高的保守性,牦牛与普通牛SRY和TRO基因这两个区域的核酸同源性分别高达99.08%和99.39%.     

海南产花鳗鲡细胞色素b基因的克隆及序列分析

线粒体细胞色素b基因是目前研究鱼类分子系统进化的重要分子标记.笔者以日本鳗鲡线粒体基因组序列为模板设计并合成引物进行PCR扩增,并将扩增产物经琼脂糖凝胶电泳和纯化后,克隆到pMD18-T载体、测序,成功得到全长为1 140 bp的海南产花鳗鲡细胞色素b(Cyt b)基因序列.将该基因全长序列递交到Genebank数据库,获得序列登录号为EF690363.用DNA分析软件MEGA2比较了海南产花鳗鲡与递交到GenBank中的8种分布在中国东南沿海、日本海域及南太平洋海域的鳗鲡属(Anguillia)细胞色素b基因的地理差异,并构建了系统进化树.结果显示:日本产和夏威夷产花鳗的地理差异小于海南产花鳗与它们之间的地理差异.     

人类基因组非冗余Exon/Intron数据库的构建

以Homo.sapiensRefSeq作为原始数据库来构建EID(Exon/Intron Database)可以克服GenBank所带来的冗余问题.通过分析RefSeq基因组数据库中每个CDS(Coding Sequence,编码序列),获得构建EID的相关的数据(基因的定义、基因标识符、基因序列、蛋白质标识符、蛋白质序列、外显子和内含子的数量、大小、总数、非翻译区(UTR)内含子、内含子相位、内含子剪切位点模式).结果表明,人类24条染色体(22条常染色体和2条性染色体,共计2 870 827355 bps)中含有32 157个基因标识符(gene blocks),其中7 398个基因为假基因,4 014个基因发生了可变剪切(Al-ternative Splicing,AS),15 533个基因含有CDS内含子,765个基因含有UTR内含子,2 585个基因不含有内含子,其他的为异常基因.     

Single gene circles in dinoflagellate chloroplast genomes.

Photosynthetic dinoflagellates are important aquatic primary producers and notorious causes of toxic 'red tides'. Typical dinoflagellate chloroplasts differ from all other plastids in having a combination of three envelope membranes and peridinin-chlorophyll a/c light-harvesting pigments. Despite evidence of a dinoflagellete satellite DNA containing chloroplast genes, previous attempts to obtain chloroplast gene sequences have been uniformly unsuccessful. Here we show that the dinoflagellate chloroplast DNA genome structure is unique. Complete sequences of chloroplast ribosomal RNA genes and seven chloroplast protein genes from the dinoflagellate Heterocapsa triquetra reveal that each is located alone on a separate minicircular chromosome: 'one gene-one circle'. The genes are the most divergent known from chloroplast genomes. Each circle has an unusual tripartite non-coding region (putative replicon origin), which is highly conserved among the nine circles through extensive gene conversion, but is very divergent between species. Several other dinoflagellate species have minicircular chloroplast genes, indicating that this type of genomic organization may have evolved in ancestral peridinean dinoflagellates. Phylogenetic analysis indicates that dinoflagellate chloroplasts are related to chromistan and red algal chloroplasts and supports their origin by secondary symbiogenesis.     

The genome sequence of Schizosaccharomyces pombe

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.     

Independent evolution of structural and coding regions in a Neurospora mitochondrial intron

The discovery of intervening sequences (introns) in eukaryotic genes has raised questions about the origin and evolution of these sequences. Hypotheses concerning these topics usually consider the intron as a unit that could be lost or gained over time, or as a region within which recombination can occur to facilitate the production of new proteins by exon shuffling. Additional complexities are observed in introns of mitochondrial and chloroplast genes which contain secondary structures required for messenger RNA splicing and open-reading frames encoding proteins. Here we describe differences in the organization of protein-coding sequences in the intron of the mitochondrial ND1 gene in two closely related species of Neurospora. These differences show that intron sequences involved in secondary structure formation and in protein coding can evolve as physically distinct elements. Indeed, the secondary structure elements of the ND1 intron can contain two different coding sequences located at two different positions within the intron.     

湿地松转录组SSR分析及EST-SSR标记开发

【目的】湿地松是南方地区优先推广的优质产脂树种。但湿地松分子遗传基础薄弱,基因组序列信息匮乏,影响了湿地松基因组学的深入研究。目前,湿地松分子研究所用的SSR标记主要来自其他近缘种或利用公共数据库中有限的基因序列资源开发的SSR标记,其多态性和通用性较差。为解决这一问题, 笔者根据湿地松转录组测序数据开发EST-SSR位点,并揭示其在转录组序列中的分布类型及特征,为湿地松分子标记辅助育种奠定基础。【方法】利用MISA软件对转录组序列进行SSRs查找和分布特征分析。查找标准参数设置为: 单核苷酸重复>10次,二核苷酸重复>6次,三、四、五、六核苷酸重复>5次。根据SSR位点两端的保守区域,利用Primer3.0设计并随机挑选120对SSR引物,通过琼脂糖电泳和毛细管电泳对来自美国和吉安的113份家系个体进行遗传多样性分析,确定引物多态性。【结果】79 574条unigenes序列中搜索到3 818个SSR位点,出现频率为4.80%,平均18.27 kb出现1个SSR位点,3 373个unigenes含有SSR位点,SSR发生频率(含SSR位点的序列数/搜索序列总数)为4.24%,其中2 980条序列含1个SSR位点,含1个以上SSR位点的序列有393条。在检测到的3 818个SSR标记中,单核苷酸分布最多,其次是二核苷酸和三核苷酸,SSR数量分别占总数的63.54%、19.15%和16.27%,而四、五、六核苷酸重复类型所占比例较小,分别为0.52%、0.13%和0.31%。SSR重复单元的重复次数分布在5~22次之间,除单核苷酸重复外的1 391个SSR中,重复5次的数量最多,为498个(35.80%);重复6次和7次的次之,分别为417个(29.98%)和198个(14.23%);重复10次以上的仅有38个(2.73%)。在检测到的731个二核苷酸重复SSR中,最常出现的重复单元为AT/AT,数量为491个(12.86%),AG/CT和AC/GT两种类型的重复单元出现的次数次之,分别为156(4.09%)和81个(2.12%)。在检测到的621个三核苷酸重复中,AAT/ATT是出现频率最高的单元,共139个(3.64%),其次是AAG/CTT,共122个(3.20%)。3 818个SSR中有24.59%的位置未知,其余的SSR则分布在非编码区域(untranslated region,UTR)或者编码区(coding sequence,CDS)上,分布数量表现为3'UTR>5'UTR>CDS。参试的120对SSR引物,有92对扩增成功(76.78%),其中24对呈现多态(20%)。24对引物(13个二核苷酸重复、7个三核苷酸重复和4个四核苷酸重复)共检测出81个等位基因,每个位点的等位基因数为2~9,平均为3.38个。多态性信息含量(polymorphism information content,PIC)为0.103~0.726,平均为0.349。【结论】通过对湿地松转录组数据的挖掘,共获得 3 818个SSR位点,主要重复单元为AT/AT和AAT/ATT,可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。基于湿地松转录组序列的SSR标记开发是可行的。     

Structure of mouse kallikrein gene family suggests a role in specific processing of biologically active peptides

The glandular kallikrein gene family comprises 25-30 highly homologous genes that encode specific proteases involved in the processing of biologically active peptides. In the mouse all the members of this family are closely linked on chromosome 7. The 9.5-kilobase nucleotide sequence of a mouse genomic clone contains one complete kallikrein gene (mGK-1), which is expressed in the male mouse submaxillary gland, and the 3' end of another (mGK-2). Differences in the coding potential of these genes and the amino acid sequences of other known kallikreins seem to be functionally related to the substrate specificity of the different enzymes.     

Polymorphism and absence of Leu-enkephalin sequences in proenkephalin genes in Xenopus laevis

The structures of the genes coding for the opioid peptide precursors proopiomelanocortin, proenkephalin (proenkephalin A) and prodynorphin (proenkephalin B), are known for some mammalian species. To gain insight into the evolutionary history of these precursors, we have examined the proenkephalin gene in the South African clawed toad, Xenopus laevis, which diverged from the principal line of vertebrate evolution some 350 Myr ago. The human proenkephalin gene consists of four exons, of which the main exon (exon IV) contains all known biologically active peptides--six Met-enkephalin sequences and one Leu-enkephalin sequence. We report here the primary structures of the putative main exons of two proenkephalin genes in X. laevis, each of which codes for seven Met-enkephalin sequences but no Leu-enkephalin, indicating that Met-enkephalin preceded Leu-enkephalin in the evolution of the proenkephalin gene. The organization of the main exons of the toad genes is remarkably similar to that of the human gene and conserved regions provide evidence for functionally significant structures. We also detect a polymorphism in one of the toad proenkephalin genes, mapping 1.5 kilobases (kb) 5' of the main exon; it is caused by an insertion/deletion of a 1-kb repetitive sequence which has the characteristics of a transposable element.     

Identification and nucleotide sequence of a diversity DNA segment (D) of immunoglobulin heavy-chain genes

A putative diversity segment of immunoglobulin heavy-chain genes (D segments) has been identified 700 base pairs 5' to JH1 DNA on the germ-line genome of the mouse. This 10-base pair D segment is flanked by two sets of sequences related to (SEE FORMULAR IN TEXT) which are possible recognition sites for a recombinase. The spacer separating the heptamer and the nonamer is 12 base pairs long on both sides of the D segment. As the space separating the two signal sequences in VH DNAs and JH DNAs is 23 +/- 1 base pairs long, the two recombinations required for creation of a complete immunoglobulin VH gene, a VH--D joining and a D--JH joining, follow a 12/23-base pair spacer rule. Allelic exclusion is discussed with respect to D segments.     

Conservation and rearrangement of mitochondrial structural gene sequences

Mitochondria contain the simplest DNA molecules that are present in eukaryotes. Mitochondrial DNA (mtDNA) is easily purified, and is an important model system for studying eukaryote gene structure and basic molecular processes. The protein sequences of mitochondrial gene products have been shown to be conserved from yeast to man, and there are definite similarities at the DNA sequence level. In contrast, the overall organization of the mitochondrial genome is drastically different in these organisms. To understand this, we need to extend work on mtDNA to a wider range of species. We have chosen to study the mtDNA of Aspergillus nidulans because a particularly comprehensive analysis of this system can be achieved using genetics as well as biochemistry, and like most eukaryotes it is an obligate aerobe, whereas Saccharomyces cerevisiae is not. We have investigated whether defined pieces of particular yeast mitochondrial genes show enough homology to Aspergillus mtDNA fragments to enable the corresponding Aspergillus genes to be located on the physical map. The results reported here show that this is the case for all five genes tested, and present the first data on the physical organization of the structural genes in the mitochondrial genome of A. nidulans.