滩羊体大品系遗传标记的研究

采用RAPD(Rondom Amplified Polymorphic DNA)技术,利用混合基因池(DNA pool)法,对滩羊体大品系、普通品系进行了DNA多态性分析,从100种具有10个碱基的随机引物中,筛选出84种引物在滩羊群体基因组中共扩增出358条带,其中22种引物的扩增产物表现为多态(占22％),且扩增出32条有差异的条带,占总带数的8.94％;62种引物的扩增产物表现为单态(占62％)。滩羊体大品系的特异性条带有5条,而普通品系的特异性条带有7条,这些特异标记可以用来鉴定滩羊的两个品系;滩羊体大品系与普通品系间的遗传距离为0.136±0.087,表明两品系之间的亲缘关系很近。     

中华绒螯蟹与日本绒螯蟹线粒体COI基因片段的序列比较研究

以相应引物PCR扩增了黄河口中华绒螯蟹线粒体细胞色素氧化酶Ⅰ亚基基因（COI）片段,PCR产物经T载体连接之后进行了克隆、测序,得到709bp的碱基序列,其A,T,G,C含量分别为34．41％,27．93％,20．03％和17．63％。并比较它与珠江流域中华绒螯蟹COI序列和日本绒螯蟹COI序列的差异,发现黄河口中华绒螯蟹与珠江 流域中华绒螯蟹COI序列完全相同,而与日本绒螯蟹差异非常明显,709或658（不计引物）位点中核苷酸差异数为32,核苷酸差异率为4．51％或4．86％（不计引物）,其中25个位点为转换,7个位点为颠换。作者倾向于支持存在中华绒螯蟹和日本绒螯蟹,或它们为同一种的两个地理亚种的观点。     

PCR引物网络与软件设计的一次成功实践

文章基于网络及BLAST,Primer Premer5等辅助软件,对苹果18SrRNA片段、ABP基因全长序列的PCR引物进行设计与探讨,完成了ABP正义表达基因和反义表达基因的扩增引物设计.结果表明,在PCR产物电泳检测的预计大小处有条带出现,测序分析证实所获得片段为目的基因.此次引物的成功设计,为相关基因克隆研究提...     

辣椒部分栽培种遗传相似性的RAPD分析

通过28个10bp的引物对30个辣椒栽培品种进行PCR扩增，共扩增出111条带，其中多态性条带有90条，占总条带数的81％，平均每个引物产生3．21个多态性条带，依据RAPD扩增结果，利用MEGA软件计算出各品种间的遗传相似性系数，构建Neighbor—Joining树形图，并结合辣椒品种的性状进行分析。结果发现：辣椒品种间的遗传相似性与辣椒果味或者果形之间总体上并无相关性，但在某些小组内，辣味品种内部或甜椒品种内部的遗传相似性高于这两种类型之间的遗传相似性；灯笼形辣椒和角形(羊角形和牛角形)辣椒品种内的遗传相似性也高于这两类品种之间的遗传相似性。试验结果为辣椒种质资源的鉴定以及育种提供了一定的参考依据。     

两个春兰变种花的AFLP遗传差异研究

采用经过优化后的DNA-AFLP分析体系,分析了春兰原变种与线叶春兰花基因组上的差异.9对引物组合在100-800bp之间共扩增出389条带.其中共有条带318条,春兰原变种有11条特异性条带,而线叶春兰有60条特异性条带.线叶春兰特异带的48.3%集中500-800bp,51.7%集中在100-500bp,春兰原变种特异带全部集中500-800bp.通过对不同引物分析发现引物对（E-AGC/M-CAT）对春兰原变种以及线叶春兰的特异性片段选择性较好.两种兰花花基因组上的差异与两种兰花花形态,以及香味差异有密切关系.通过对两种兰花花基因组的AFLP遗传差异的探讨,为今后研究兰花花香基因,兰花育种奠定了一定理论基础.     

串联表达载体中短重复序列(BmKb1)_n模板的PCR效率及其优化条件研究

采用Overlap PCR法获取BmKb1基因,构建载体pGEX-6p-1/BmKb1,同尾酶技术用于构建串联表达载体pGEX-6p-1/(BmKb1)n(n=2,4,6,8,10)。以pGEX-6p-1/(BmKb1)n为模板,使用标准PCR程序研究短重复序列PCR效率;以BmKb1八倍体为模板,分别研究PCR循环数、引物终浓度及退火温度对短重复序列PCR效率及特异性的影响。结果表明:串联重复序列数目增加,则非特异性条带增加,特异性条带含量减少;BmKb1基因串联体PCR扩增在18～20循环数、引物终浓度为0.05～0.1μM、60～62℃较高退火温度时,可获得相对高产量高特异性BmKb1基因串联体PCR片段。本研究将为串联表达载体克隆、筛选、测序及基因组STRs序列克隆测序提供技术参考。     

一个在中华绒螯蟹性腺中特异表达的Sox基因HMG盒区的克隆与鉴定

根据人的睾丸决定因子（SRY）的高速泳动组蛋白区设计简并引物,以中华绒螯蟹雌雄个体的基因组DNA为模板进行PCR扩增,从雌雄体均扩出220bp左右的条带,说明中华绒螯蟹中存在SRY基因的同源体,且无性别差异．经克隆、测序后得到两条SRY盒区相关基因（Sox基因）片段,ES1和ES2,其序列与人相应Sox基因保守区的最高同源性分别为66％和93％．ES1和ES2在成体8种组织中的检测结果表明,ES1在精巢、肌肉、心脏、肝脏、鳃、胸神经团、不同时期的卵巢及排出的成熟卵等组织中均表达,而ES2只在精巢、成熟的卵巢以及排出的成熟卵中有表达,说明ES2基因可能参与性腺的发育和早期胚胎发育过程．     

蔬菜保护地土壤木霉菌的UP-PCR多样性

为明确蔬菜保护地土壤中木霉菌的遗传多样性,采用通用引物PCR对蔬菜保护地部分土壤样品中分离到的木霉菌进行了多样性分析,并用UPMGA法对供试菌株的扩增片段数据进行图谱聚类分析。从13条通用引物中筛选出5条引物,分别为3-2,AS4,AS15 inv,AS19和L45;扩增出46条谱带,其中多态性条带43条,占总条带数的93.5%。当相似系数为0.74时,可将24个菌株划分为9个组,分别为A1,A2,B1,B2,B3,B4,B5,C1和C2。     

坛紫菜种质资源遗传多样性的AFLP分析

利用AFLP技术从基因组DNA水平上分析福建省主要坛紫菜栽培品系和诱变筛选获得的突变品系的遗传差异,在25对引物中,有11对能得到重复性好的多态性扩增条带,总共在667个位点能扩增出条带,每对引物扩增带数为24~90条,并且各对引物扩增结果均显示样品间存在差异.共得到522条多态性条带,占总扩增带的78.3%.根据AFLP图谱计算了不同样品间的遗传距离(D)和相似性系数(GS).优良品系GL和CCS之间的遗传距离最小,仅为0.264,不同样品间的相似性系数介于0.602~0.736之间.并根据D值绘制了它们的UPGMA聚类图谱.     

应用RAPD技术对新疆布顿大麦遗传多样性的研究

利用RAPD分子标记，通过40条10碱基随机引流，对32份新疆布顿大麦进行了遗传多样性评价。在40个10碱基随机引物中，有22个引物（占55％）的扩增产物具有多态性，每条多态性引物能产生1－7条多态性DNA带纹。40个引物共扩增227条带，其中95条（占41．9％）具有多态性，平均每条引物能产生1．7条多态性DNA带纹。基于RAPD技术计算了32份布屯大麦间的遗传相似系数（GS）变化值为0．427－0．848，平均值为0．681，利用不完全加权算术平均数（UPGMA），采用1－GS矩阵对其进行遗传聚类，结果表明，RAPD标记将32份参试材料区分为3类（或亚类），来源于新疆北部的20份材料有17份材料（占85％）聚入Ib亚类，而第Ia亚类则主要来自于新疆中部的一些材料。研究表明，RAPD标记揭示的新疆布顿大麦间的遗传多样性与其地理来源紧密相关。     

声波刺激对拟南芥基因表达的影响

运用银染mRNA逆转录差异显示(DDRT-PCR)技术,初步研究了声波刺激对拟南芥差异基因表达的影响.研究分离得到SA3、SG2-1、SG2-2、SG7-1、SG7-2、CA2共6个差异表达基因片段,进一步运用Northern点杂交确定SA3、CA2、SG7-1为阳性片段,其中SA3片段属于声波刺激特异表达基因, SG7-1片段属于声波刺激增强表达基因,CA2片段属于声波刺激抑制表达基因.研究结果表明植物在基因水平对声波刺激具有响应,为下一步探索植物响应声波应力的分子生物学调控机理奠定了基础.     

一种优化的mRNA差异显示技术分析白刺盐碱胁迫表达相关基因研究

mRNA差别显示PCR技术是分离、检测差异基因表达的有力工具 我们研究了存在的可能影响DDRT-PCR研究结果的因素 通过改变DDRT-PCR方法中的引物、PCR反应中的退火温度等条件,得到了一种优化的DDRT-PCR技术,运用该项技术研究了白刺盐碱胁迫相关基因的差异表达 结果发现,运用这种方法得到的差异表达基因片段假阳性率较低,且差别条带较长,多为800bp以上,克服了DDRT-PCR技术中的主要问题,为更广泛地运用该项技术奠定了基础     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

Identification of genes induced during Medicago sativa nodule development by using the cDNA-AFLP technique

Under limited nitrogen conditions, rhizobia are ableto induce the formation of nitrogen-fixing root nodules on their leguminous plant host. This organogenetic process is triggered by a complex exchange of molecu- lar signals between the host plant and bac…     

Identification of differentially expressed genes in esophageal cancer through SSH in combination with high throughput reverse Northern screening

To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.     

Cloning and identification of a novel human glioma differentiation related gene GDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed gene GDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame of GDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows that GDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns of GDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

Cloning and identification of a novel human glioma differentiation related geneGDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

Cloning and expression analysis of MBLL cDNA

The mbl (muscleblind) gene of Drosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation of mbl gene will disturb the differentiation of all the Drosophila's photoreceptors. Primers have been designed according to human EST086139, which is highly homologous to mbl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designated MBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology to Drosophila's mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show that MBLL is a widely expressed gene, but the expression amounts differ in these tissues.