水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Xa-4紧密连锁的分子标记M55的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片DNA、半粒种子提取物及Xa-4基因的杂合体DNA的PCR特异扩增,初步建立了Xa-4的PCR标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Xa-4在这些材料中的分布情况。 Abstract　Based on the sequence of a DNA marker tightly linked to the rice bacterial blight（BB） resistance gene Xa-4, two primers were designated and synthesized to develop a PCR marker for the gene. Specific amplified polymorphism analysis was carried out with these primers on a set of BB resistance isogenic lines and pyramided lines developed by IRRI. Two PCR bands were revealed corresponding to lines with dominant Xa-4 and those with the recessive allele, respectively, regardless the lines pyramided with other resistance genes. A hybrid with heterozygous Xa-4 produced both of the two allele PCR pattern. Then, the PCR marker was used to survey a range of hybrid rice germplasm. The results of the germplasm survey will be useful in hybrid rice breeding programs aimed at exploiting Xa-4.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

用交错延伸剪接PCR技术扩增油菜花粉育性基因MS2Bnap全长cDNA序列

交错延伸剪接PCR是一种用于分子进化研究的DNA改组技术.本实验利用其原理,将已获得的油菜花粉育性基因MS2Bnap有部分序列重叠的三段PCR产物作为模板进行扩增,获得了花粉育性基因MS2Bnap的全长cDNA序列。 Abstract:SOE by PCR is one of DNA shuffling technologies for molecular evolution engineering.Using the theory of SOE by PCR,the full-length cDNA sequence of pollen fertility gene MS2Bnap of Brasscia napus was amplified with three obtained fragments as templates which have part overlapping of MS2Bnap sequence.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

Quantum dots （QDs） are of great interest due to their unique chemical and physical properties. Recently, a hot start （HS） polymerase chain reaction （PCR） amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high,fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was preincubated for 2 h at 50℃. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incu- bation at 50℃ before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA poly- merases.     

Co-expression and immunity of Legionella pneumophila mip gene and immunoadjuvant ctxB gene

The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplifiedby PCR respectively.The amplified cDNA was ligated to the pcDNA3.1(+)vector.The recombinant plasmidspcDNA3,1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR,and further confirmedby sequencing analysis.NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB accordingto the Lipofection method.Transient and stable products of the co-expression of the mip gene and ctxB genewere detected by immunofluorescence and Western blotting.The results showed that NIH3T3 cells weresuccessfully transfected,and that the transiently and stably co-expressed products can be detected in thetransfected cells.To detect the humoral and cellular immune response in immunized mice induced by the co-immunization of the mip and ctxB genes,female BALB/c mice were immunized intramuscularly with pcDNA3.1-mip and pcDNA3.1-ctxB.The results showed that the specific antibody titer and the cytotoxic T-lymphocyteresponse for pcDNA3,1-mip immunization and co-immunization were increased compared with that ofpcDNA3.1(+) immunization.Furthermore,the specific antibody titer and cytotoxic T-lymphocyte responsefor co-immunization were increased compared with that of pcDNA3.1-mip immunization.Statistical analysisusing one-way analysis of variance(ANOVA)showed that there was a significant difference between thegroups(P<0.01).The results indicated that the ctxB gene enhanced the humoral and cellular immune responseto the mip gene immunization.These findings provide experimental evidence to support the development ofthe L.pneumophila DNA vaccine.     

In situ amplification of DNA fragments specific for human Y chromosome in cellular nuclei by PCR

Using single primer pairs Y3 and Y4, in siru polymerase chain reaction (in situ PCR) was successfully performed on the specimen slides of peripheral leukocytes. By both of the direct digpxiginin-11-dUTP incorporation into PCR products with in situ PCR (direct in situ PCR) and in situ PCR followed by detection of in situ hybridization (indirect in siru PCR), DNA fragments specific for human Y chromosome were obviously amplified in cellular nuclei of specimens on the slides. The results were verified by Southern analysis. The methodology of in situ PCR and its application were discussed.     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.     

Detection of specific DNA from crude extracts of rice seed grains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

To rapidly detect specific genes, crude extracts prepared from rice seed grains were used as templates for PCR, the PCR products were digested with restriction enzymes or urasil-DNA glycosylase, and then matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) was used to detect amplified DNA. It was possible to amplify small DNA fragments (50–60 bp), but not large ones (>200 bp), using crude extracts as the PCR template. This method can be completed within 1 h, including extractions, and is well suited to automation for high-throughput analyses.     

竞争性RT-PCR法及对大肠杆菌acrA基因mRNA表达水平的定量研究

建立了用于检测大肠杆菌(Escherichia coli)ATCC25922的acrA基因mRNA表达水平的定量竞争性RT-PCR(QC-RT-PCR)体系。PCR合成目标片段的突变型片段(321bp)作为内标准模板(Internal standard,IS),与目标片段一致的片段(389bp)作为目标模板(Target standard,TS),优化两种模板共扩增体系;梯度稀释IS与等量大肠杆菌cDNA样本共扩增,扫描电泳条带,软件分析数据。结果表明,引物设计合适,以IS和TS为模板实现共扩增,产物(321bp和389bp)通过1.5%琼脂糖凝胶电泳有效分离;梯度稀释IS与cDNA共扩增产物出现亮度梯度电泳条带;获得一元回归曲线y=-0.345+0.097x(相关系数r=0.959,标准差s=0.05997)。该研究成功构建内标准模板,优化的共扩增PCR体系实现了对大肠杆菌ATCC25922中acrA基因mRNA表达水平的检测,具有简便、高效、敏感度高等优点。     

Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.

The PCR is used widely for the study of rRNA genes amplified from mixed microbial populations. These studies resemble quantitative applications of PCR in that the templates are mixtures of homologs and the relative abundance of amplicons is thought to provide some measure of the gene ratios in the starting mixture. Although such studies have established the presence of novel rRNA genes in many natural ecosystems, inferences about gene abundance have been limited by uncertainties about the relative efficiency of gene amplification in the PCR. To address this question, three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative. The PCR products were digested with restriction endonucleases, and the frequencies of genes in the products were determined by electrophoresis on an Applied Biosystems 373A automated DNA sequencer in Genescan mode. Mixtures of two templates amplified with the 519F-1406R primer pair yielded products in the predicted proportions. A second primer pair (27F-338R) resulted in strong bias towards 1:1 mixtures of genes in final products, regardless of the initial proportions of the templates. This bias was strongly dependent on the number of cycles of replication. The results fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids.     

Effects of temperature, incubation period and substrate on production of fusaproliferin by Fusarium subglutinans ITEM 2404

The kinetics of the production of fusaproliferin by Fusarium subglutinans ITEM 2404 in maize and rice cultures was investigated at various incubation temperatures. The growth rate of F. subglutinans was highest at 20 degrees C and 25 degrees C in maize cultures and at 15 degrees C in rice cultures. Although the growth rate was higher in rice than in maize, the maximal production of fusaproliferin was obtained in maize cultures, with a maximum yield (4309 microg g(-1)) at 20 degrees C for 6 weeks. In rice cultures the optimal incubation regimen was at 15 degrees C for 6 weeks, with a fusaproliferin level of 1557 microg g(-1). The production of fusaproliferin at 25 degrees C and 30 degrees C in both substrates was very low, with maximal yield at 25 degrees C of 979 microg g(-1) after 2 weeks and 143 microg g(-1) after 3 weeks in maize and rice cultures, respectively.     

以HIV-1 5＇LTR为靶点的药物筛选细胞模型的构建

构建以人类免疫缺陷病毒（HIV-1）5＇LTR为靶点的药物筛选细胞模型,用于体外筛选潜在的对HIV-1启动子具有抑制作用的药物,或用于筛选与HIV-1复制相关的宿主因子。通过PCR扩增HIV-1 5＇LTR片段和胸苷激酶基因（thymidine kinase gene,TK基因）,以这2片段为模板进行重叠PCR将两者连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系;加入药物更昔洛韦（GCV）检测TK基因的表达。成功构建了HIV-1 5＇LTR调控TK基因表达的稳定细胞系,在其培养过程中加入药物更昔洛韦时,细胞逐渐死亡。构建了以HIV-1 5＇LTR为靶点的药物筛选细胞模型,该模型利用TK基因作为报告基因方便灵敏,可用于筛选针对HIV-1 5＇LTR的潜在抗HIV药物。     

红松RAPD实验中各组成成份含量对实验结果的影响

本文采用Ｐｒｏｍｅｇａ的ＰＣＲ反应系统,以从红松幼叶中提取的总ＤＮＡ为模板,进行随机扩增多态ＤＮＡ（ＲＡＰＤ）实验。     

一种快速高效获取丝状真菌PCR反应模板的方法

建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。     

Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis

DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious. In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates, named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Ｘａ－４紧密连锁的分子标记Ｍ５５的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片ＤＮＡ、半粒种子提取物及Ｘａ－４基因的杂合体ＤＮＡ的ＰＣＲ特异扩增,初步建立了Ｘａ－４的ＰＣＲ标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Ｘａ－４在这些材料中的分布情况。