从Cytb基因序列探讨蝽类部分昆虫的系统发生

通过对蝽总科18种昆虫线粒体DNA细胞色素6基因部分序列进行PCR扩增和序列测定,比较其同源性,统计密码子使用频率并应用生物学软件构建系统发育树．在获得的432bp序列中,碱基T,C,A和G的平均含量分别为37．4％,18．8％,31．8％和12．0％,表现出强烈的AT偏向性．就每个氨基酸密码子采看,第3位点的A＋T含量较高,达到83．6％．谊序列片段中共有215个核苷酸位点发生变异（约占49．8％）,种间变异较大．在氨基酸组成上,共编码144个氨基酸。其中有47个发生变异。变异率为32．7％．碱基替换主要发生在密码子第3位点,转换略多于颠换．以筛豆龟蝽（Megacopta cribraria）为外群构建的系统发育树支持荔蝽、盾蝽作为独立的科与蝽科并列,滴蝽从蝽亚科划出归属于舌盾蝽亚科,但蝽科内各亚科间及蝽亚科各族间的系统进化关系与传统的形态分类结果不完全一致,还有待于进一步研究．     

绵羊角蛋白关联蛋白KAP6-1基因5'端调控区的分子克隆及测序结果比较

根据绵羊毛囊角蛋白关联蛋白（KAP6-1）基因已知DNA序列设计合成了两个特异性引物,以绵羊基因组DNA为模板,PCR扩增出1042bp的特异性片段,连接到pMD19T载体中获得该片段克隆．经过快速提质粒法筛选、限制性内切酶分析表明该克隆包含所需的目的片段．DNA测序结果也证明该克隆片段与原基因5＇端调控区序列相比具有很高的一致性．研究结果为今后制备转基因克隆动物,在毛囊细胞中特异性表达绒毛生长调控基因奠定了基础．     

耐热碱性磷酸酯酶基因的DNA序列分析

从栖热菌中克隆到产耐热碱性磷酸酯酶（FD－TAP）基因并进行了DNA序列分析,结果表明此2．0kb的片段含有一个1056bp的开放阅读框,编码501个氨基酸的蛋白质,其N端有一26个氨基酸的信号肽．在起始密码子的上游5bp处有一个5＇－GGAGGT－3＇的SD序列．基因编码区的（G＋C）％为68．7％,第3位密码子（G＋C）％为92．7％．FD－TAP的氨基酸序列与大肠杆菌等生物的碱性磷酸酯酶氨基酸序列比较,相同性为27％,相似性为38％．中央β－折叠区及与活性中心相关的氨基酸残基高度保守．表明FD－TAP具有与大肠杆菌碱性磷酸酯酶相似的结构和作用机制．在相当于大肠杆菌碱性磷酸酯酶的His370至His412两个金属离子结合部位之间,FD－TAP有一72个氨基酸的插入片段,提示该插入片段与FD－TAP的高耐热性相关．     

大肠杆菌的同义密码子使用偏性对蛋白质折叠速率的影响

考虑到同义密码子使用偏性的物种差异性,选取大肠杆菌的54条核蛋白为研究对象,将每条核蛋白按二级结构截取出α螺旋片段、β折叠片段和无规卷曲(α-β混合)片段,然后找到每个片段相关的核酸序列信息,计算其同义密码子使用度和相应肽链的折叠速率.在此基础上,分析了同义密码子使用偏性和相应肽链折叠速率之间的相关性,发现对于不同二级结构类的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关.因此,在蛋白质折叠中,同义密码子的使用偏性起着重要作用.     

基于模糊聚类算法的DNA序列分类模型

通过分析DNA序列链之间的关联程度,构造出模糊相似矩阵,计算出传递闭包,获得详细的动态聚类过程.使用该模型的优点是,输入少量参数及阈值,就可以较准确的对DNA序列的集合进行分类.验证该算法的有效性.     

甘蓝型油菜乙醇酸氧化酶(GO)基因片段的克隆及hpRNA表达载体构建

通过PCR从甘蓝型油菜(Brassica napus)华双4号基因组DNA中扩增出乙醇酸氧化酶(GO)基因片段,DNA序列分析表明扩增GO基因片段的外显子部分与报道序列相同.以扩增出的GO基因片段作模板从一端设计引物扩增出一个相应的小片段.将GO基因大小两个片段反向连接,插入到植物表达载体2300-nap的napin启动子和nos终止子之间,植物表达载体2300-35S的35S启动子和nos终止子之间,分别构建成可转录表达出发夹RNA(hairpin RNA,hpRNA)结构的种子特异型和组成型油菜hpRNA干扰载体.     

分离与克隆中国人TPO基因片段及序列分析

应用ＲＴＰＣＲ技术从中国人胎肝细胞中分离出１个５６６ｂｐ大小的基因片段,经过克隆、限制性内切酶鉴定和序列分析证实为ＴＰＯ的ｃＤＮＡ片段．与ＧｅｎＢａｎｋ中发表的人ＴＰＯｍＲＮＡ的序列比较同源性在８２％～９９％之间,仅有２个碱基不相同,在１７５位密码子（５２３～５２５位的碱基）分别是ＣＧＧ和ＣＡＡ,即精氨酸（Ｒ）的位置上,中国人是谷胺酰氨（Ｇ）．而与测得的韩国人序列比较,在相应位置的氨基酸是相同的     

盐生盐藻肌动蛋白基因及其5′上游片段的克隆

在盐生盐藻 c DNA文库和基因组文库中对肌动蛋白基因进行了筛选 ,结果得到了一个c DNA克隆和两个基因克隆 ,并进一步从基因克隆中克隆出该基因的 5′上游片段 .检索结果表明 ,所得到的 c DNA和基因属于盐生盐藻的肌动蛋白基因 .通过将盐生盐藻肌动蛋白基因序列同其c DNA序列及其他生物的肌动蛋白序列相比较 ,给出了一个此基因 5′上游区大概的结构     

苦瓜(Momordica Charantia L.) MADS-box基因BAG启动子的克隆分析及表达载体的构建

作者提取苦瓜基因组总DNA，构建了DNA步移文库，并根据已克隆并公布的苦瓜MADS-box基因BAG的基因序列设计引物，通过染色体步移技术克隆出BAG基因起始密码子上游调控序列BAGP．对BAGP的鉴定和分析表明其具备大多数高等植物启动子的保守元件，预测它对BAG基因的表达具有一定的作用．为鉴定BAG基因的基本启动子元件。将基因5′侧翼序列做缺失片段分析，利用PCR方法从BAGP中得到3个大小不等两端带有HindⅢ,BamHⅠ酶切位点的片段BAGPl，BAGP2和BAGP3，定向插入载体pMGFP4(pBI221改建，报告基因为GFP)中，取代原有的CaMV35S启动子，构建了由驱动报告基因GFP的植物表达裁体BAGPVl，BAGPV2和BAGPV3．     

酵母密码子编码的α降钙素基因相关肽基因的化学合成和克隆

记叙了α降钙素基因相关肽基因的化学合成和克隆.该多肽的合成基因序列是根据已知的氨基酸序列,选用酵母偏爱的密码子,由计算机辅助设计而成的,共136个碱基对.全基因分为六个寡核苷酸片段在DNA合成仪上合成.这些片段混合退火后,用T4DNA连接酶一次装配成功,并克隆到M13mp18载体上.经核酸序列分析,证明所得克隆的核苷酸顺序与设计完全一致.     

A DNA sequence alignment algorithm using quality information and a fuzzy inference method

DNA sequence alignment algorithms in computational molecular biology have been improved by diverse methods. In this paper, we propose a DNA sequence alignment that uses quality information and a fuzzy inference method developed based on the characteristics of DNA fragments and a fuzzy logic system in order to improve conventional DNA sequence alignment methods that uses DNA sequence quality information. In conventional algorithms, DNA sequence alignment scores are calculated by the global sequence alignment algo- rithm proposed by Needleman-Wunsch, which is established by using quality information of each DNA fragment. However, there may be errors in the process of calculating DNA sequence alignment scores when the quality of DNA fragment tips is low, because only the overall DNA sequence quality information are used. In our proposed method, an exact DNA sequence alignment can be achieved in spite of the low quality of DNA fragment tips by improvement of conventional algorithms using quality information. Mapping score param- eters used to calculate DNA sequence alignment scores are dynamically adjusted by the fuzzy logic system utilizing lengths of DNA fragments and frequencies of low quality DNA bases in the fragments. From the experiments by applying real genome data of National Center for Biotechnology Information, we could see that the proposed method is more efficient than conventional algorithms.     

A DNA sequence alignment algorithm using quality information and a fuzzy inference method

DNA sequence alignment algorithms in computational molecular biology have been improved by diverse methods. In this paper, we propose a DNA sequence alignment that uses quality information and a fuzzy inference method developed based on characteristics of DNA fragments and a fuzzy logic system in order to improve conventional DNA sequence alignment methods that uses DNA sequence quality information. In conventional algorithms, DNA sequence alignment scores are calculated by the global sequence alignment algorithm proposed by Needleman-Wunsch, which is established by using quality information of each DNA fragment. However, there may be errors in the process of calculating DNA sequence alignment scores when the quality of DNA fragment tips is low, because only overall DNA sequence quality information are used. In our proposed method, an exact DNA sequence alignment can be achieved in spite of low quality of DNA fragment tips by improvement of conventional algorithms using quality information. Mapping score parameters used to calculate DNA sequence alignment scores are dynamically adjusted by the fuzzy logic system utilizing lengths of DNA fragments and frequencies of low quality DNA bases in the fragments. From the experiments by applying real genome data of National Center for Biotechnology Information, we could see that the proposed method is more efficient than conventional algorithms.     

用加端PCR技术改造h－IGF－1cDNA

用ＤＮＡ合成仪合成了分别带有ＰｓｔＩ位点和ＳａｌＩ位点及终止密码子的２个用于扩增ｈＩＧＦ－１ｃＤＮＡ的ＰＣＲ引物．利用合成的引物，７００ｂｐ长的ｈＩＧＦ－１ｃＤＮＡ模板和Ｔａｑ聚合酶进行ＰＣＲ扩增．扩增产物经电泳鉴定后克隆进Ｍ１３ｍｐ１８载体，进行核苷酸序列分析．结果显示：ＰＣＲ产物含已发表的ｈＩＧＦ－１成熟蛋白的编码序列和５＇端的ＰＳｔＩ位点及３＇端的ＳａｉＩ位点及终止密码ＴＡＧ．用加端ＰＣＲ技术成功地扩增和改造了ｈＩＧＦ－１的编码序列．     

Cloning and analysis of the antagonistic related genes of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> B8

To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

大肠杆菌高效表达载体pSY621的构建

基因在大肠杆菌中的表达往往受转录起始区的二级结构所影响,通过改变原有载体ｐＳＹ６００的ＴＩＲ区域的序列,来改造出一个能高效表达的载体ｐＳＹ６２１。在表达载体上接入ＡＮＧ（Ａｎｇｉｏｇｅｎｉｎ）,ＮＴ４（Ｎｅｕｒｏｔｒｏｐｈｉｎ－４）基因,由ＰＣＧＥＮＥ软件来计算二级结构和自由能,设计修改片段,产生新载体ｐＳＹ６２１。通过表达ＡＮＧ,ＮＴ４,ｐＺＰ３（ｐｉｇ ｚｏｎａ ｐｅｌｌｕｃｉｄａ）基因来检测     

Variation and Characterization Analysis of Partial Fragment of Fibroin Gene From Silkworm, Antheraea pernyi

A 1.4Kb DNA fragment containing 3‘ flanking sequence of fibroin gene of silkworm, Antheraea perny/, was obtained from the silk gland‘s mRNA of 5th larva. Analysis of this sequence with another A. pemyi fibroin protein (accession No. 1383241) revealed that it consists of a completely open reading frame (ORF), which includes 14 polyalanine-containing units (motifs) and 100bp 3‘-UTR. The sequence of the predicted amino acid reveals the highest level of overall iden-tity (90%) with 1383241. It was found that it loses a repeat region at the upstream of TAA codon and some mutations. A putative polyadenylation signal AATAAA tail was found in position 1300, which follows the termination codon.     

人尿激酶原在大肠杆菌中表达的研究

用限制性内切酶将人尿激酶原cDNA酶切成一系列基因片段,并分别在大肠杆菌中表达。发现其中1个富含大肠杆菌稀有密码子AGG(精氨酸)的片段表达量低,成为人尿激酶原cDNA在E.coli中高效表达的限制因素。将富含AGG(精氨酸)的片段在E.coliBL21-CodonPlus^TM-RIL中表达,通过该菌株引入dnaY基因(即tRNA_agg/aga(Arg))使该片段的表达量提高了10倍。最后用同样的方法提高全长人尿激酶原cDNA在大肠杆菌中的表达量,使其表达量达到全菌蛋白的5%。     

基于多阶段的模糊C-均值算法的模糊聚类分析研究

对模糊聚类分析算法进行研究,在模糊Ｃ－ 均值算法（ＦＣＭ）的基础上加以改进,将聚类过程分为二个阶段,形成多阶段模糊Ｃ－ 均值算法（ＭＦＣＭ）,使其对Ｉｒｉｓ数据聚类．研究表明：多阶段的模糊Ｃ－ 均值算法比模糊Ｃ－ 均值算法性能优越．     

PSO高斯诱导核模糊c均值聚类算法*

为了避免陷入梯度法局部极值以提升模糊聚类算法聚类性能,提出PSO高斯诱导核模糊c均值聚类算法(PSO Gauss-induced kernel fuzzy c-means clustering algorithm, PSO-GIKFCM)。首先将高斯核函数应用于模糊c聚类算法(FCM)目标函数,得到高斯核模糊聚类目标函数。然后在高斯核特征空间和输入空间利用梯度法得到两空间聚类中心,将特征空间聚类中心与样本的内积核矩阵代入输入空间聚类中心,从而得到高斯诱导核的聚类中心。最后在解空间利用粒子群算法(PSO)对模糊隶属度进行寻优估计,并结合目标函数和聚类中心构成PSO-GIKFCM参数估计迭代流程。PSO-GIKFCM算法基于粒子群算法保证其收敛性,聚类中心仅为模糊隶属度的函数,PSO生物进化算法在解空间全局寻找优解,且将模糊指标扩展为大于0的情况。通过仿真实验验证了所提出算法的有效性。