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。     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNA Bac-N-Blue^TM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNA Bac-N-Blue^TM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。     

拟南芥AtNHX2启动子的克隆及表达模式分析(英)

AtNHX2基因是拟南芥NHX基因家族的一员,编码了一种液泡膜中的Na⁺/H⁺反向运输体并对拟南芥的耐盐能力起着重要的作用.采用PCR扩增的方法克隆了拟南芥AtNHX2基因启始密码子上游约2.8 kb的DNA片段,并将其克隆到植物表达载体pCAMBIA1301-1中,通过基因枪轰击洋葱表皮瞬时表达的方法,初步检测启动子的活性.将重组质粒pCAMBIA1301-1/AtNHX2 promoter转化拟南芥并筛选纯合子.AtNHX2 promoter-GUS分析显示AtNHX2在所有的组织中均有表达,包括根尖.在保卫细胞中检测到了强烈的GUS表达,这一结果表明,AtNHX2对特殊细胞的pH调控和K⁺自身稳定方面起着重要的作用.AtNHX2启动子的活性可被NaCl抑制,并且抑制的强度和NaCl的浓度成正相关. 300 mmol/L KCl处理可增强启动子的活性,说明NaCl和KCl是在转录水平上调控AtNHX2的表达.在老叶中GUS活性比在新叶中GUS活性强,这说明了AtNHX2优先将有毒的离子积累在老叶中,从而有利于植物的正常发育.在根毛细胞中也观测到了强烈的GUS活性,这就暗示了AtNHX2在扩大的液泡中储存Na⁺.     

用基因芯片检测DPYD等位基因在受试人群中的发生频率

二氢嘧啶脱氢酶基因（DPYD基因）所编码的二氢嘧啶脱氢酶（DPD酶）是氟化嘧啶类抗肿瘤药物代谢的主要限速酶,其活性存在显著的个体差异,并因此影响药物的疗效和毒副作用.大部分编码低/无活性酶的突变型等位基因是由于基因中的单核苷酸多态性（single nucleotide polymorphism,SNP）造成的,检测这些SNPs是预测患者对药物的反应和实现个体化给药方案的基础.制备并优化了用于检测DPYD基因中6个已知SNPs所编码的等位基因（DPYD^*2,^*3,^*4,^*5,^*9,^*12）的基因芯片,建立了该芯片的基因分型标准.并利用该芯片检测了肿瘤患者（112例）、肾病患者（83例）和健康者（45例）中DPYD突变型等位基因的发生频率.在受试人群中,突变型等位基因DPYD^*5和DPYD^*9平均发生率分别为32.08%和11.25%,未发现DPYD^*2,^*3,^*4,^*12突变型等位基因.而且以上单碱基突变的发生率在肿瘤患者、肾病患者和健康者间以及男性、女性肿瘤患者间无显著性差异,表明其与疾病的发生或性别无显著性关联.对20例标本的基因分型结果采用直接测序法进行验证,19例基因芯片分型结果与直接测序法结果相一致.DPYD^*5、DPYD^*9突变型等位基因在受试人群中具有较高的发生率.利用基因芯片能够对其实现快速准确的检测.     

通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究

用PCR方法从海洋单细胞蓝藻聚球藻7002（Synechococcus sp. PCC 7002）基因组DNA中扩增得到藻蓝蛋白β亚基基因（cpcβ）的上游序列（Pcpcβ）,及编码谷氨酰胺合成酶的glnA基因片段．以Pcpcβ作为启动子、以glnA基因片段作为整合平台,构建含有小鼠金属硫蛋白-Ⅰ（mMT-Ⅰ）cDNA的同源整合表达载体pKGC-MT.通过自然转化法将整合表达载体导入聚球藻7002中,经氨苄青霉素筛选,得到遗传性状稳定的转基因藻.PCR检测证明mMT-Ⅰ基因已整合到蓝藻基因组DNA上;蛋白质印迹表明mMT-Ⅰ已在蓝藻中表达;ELISA结果显示mMT-Ⅰ在蓝藻中的表达量约为800 μg/g.     

西瓜花叶病毒HC-Pro基因在毕赤酵母中的分泌表达与功能研究

利用RT-PCR方法扩增出西瓜花叶病毒HC-Pro的基因,长度为1371bp,并构建了真核表达质粒pPIC9K-WHC。将重组质粒经SalⅠ单酶切后电转化Pichia pastoris GS115菌株,经PCR鉴定与G418、MD和MM培养基筛选,获得Mut⁺/His⁺表型高拷贝转化子。经1%甲醇诱导5d后,SDS-PAGE检测发酵液上清,在66kD处有一特异蛋白条带表达。Western blot 鉴定表明,表达蛋白可以和HC-Pro蛋白抗血清发生结合反应。Far-Western blot 证明该蛋白能与西瓜花叶病毒CP蛋白结合,支持了HC-Pro蛋白协助传毒的“桥梁”学说。     

西瓜花叶病毒HC-Pro基因在毕赤酵母中的分泌表达与功能研究

利用RT-PCR方法扩增出西瓜花叶病毒HC-Pro的基因,长度为1371bp,并构建了真核表达质粒pPIC9K-WHC。将重组质粒经SalⅠ单酶切后电转化Pichia pastoris GS115菌株,经PCR鉴定与G418、MD和MM培养基筛选,获得Mut⁺/His⁺表型高拷贝转化子。经1%甲醇诱导5d后,SDS-PAGE检测发酵液上清,在66kD处有一特异蛋白条带表达。Western blot 鉴定表明,表达蛋白可以和HC-Pro蛋白抗血清发生结合反应。Far-Western blot 证明该蛋白能与西瓜花叶病毒CP蛋白结合,支持了HC-Pro蛋白协助传毒的“桥梁”学说。     

肿瘤中neu基因的过量表达及其抑制

neu基因编码一种和表皮生长因子受体同源的磷酸蛋白,具有酪氨酸激酶的活性.近年来在多种人类肿瘤中发现neu基因的扩增和(或)过量表达.一些蛋白质因子或化学药物可以在转录水平阻遏neu基因的过量表达或者降低其产物p185^neu的酪氨酸激酶活性,抑制具有neu基因过量表达的癌细胞的转移和增殖.     

Rapid amplification of genomic DNA ends bynla III partial digestion and polynucleotide tailing

We report a simple and efficient method, which combines restriction endonuclease digestion and deoxynucleotide tailing, for cloning unknown genomic sequences adjacent to a known sequence. Total genomic DNA is partially digested with the frequent-cutting restriction enzymeNla III. A homo-oligomeric cytosine tail is added by terminal transferase. The tailed DNA fragments are used as the template for cloning flanking regions from all sequences of interest. A first round PCR amplification is performed with a gene-specific primer and the selective (modified polyguanine) anchor primer complementary to the cytosine tail and theNla III recognition site, with a universal amplification primer sequence at its 5′ end. This is followed by another PCR amplification with a nested gene-specific primer and the universal amplification primer. Finally, the amplified products are fractionated, cloned, and sequenced. Using this method, we cloned the upstream region of a salt-induced gene based upon a partial cDNA clone (RSC5-U) obtained from sunflower (Helianthus annuus L.).     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

PCR-RFLP of S locus for identification of breeding lines in cruciferous vegetables

A simple method of detecting polymorphism of S locus glycoprotein gene, SLG, in Chinese cabbage and cabbage was developed, and used for identification of breeding lines. DNA was amplified by the polymerase chain reaction (PCR) with a pair of primers having S ₆ SLG sequences from inbred lines, and digested with restriction endonucleases which recognize tetranucleotide sequences. The cleaved DNA fragments were size-fractionated by polyacrylamide gel electrophoresis and detected by silver staining. PCR with S ₆ SLG primers amplified a fragment of ca. 1.3kb in more than half of the inbred lines tested. After digestion, polyacrylamide gel electrophoresis revealed polymorphism between the amplified 1.3kb DNA fragments. These polymorphic bands were detected by Southern hybridization using a probe of S ₆ SLG cDNA, suggesting that the amplified DNA was SLG. Primers having the SLG sequences of S ₂ , a representative of recessive S alleles, were used for amplification of SLG in the lines which did not give the 1.3kb DNA fragment by the PCR with S ₆ SLG primers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariant SLR-2 sequence of 1.3kb in addition to the polymorphic S ₂ SLG related sequences. Although the used primer sequences still need improvement for the analysis of recessive S alleles, PCR-RFLP of SLG was considered to be useful for identification of breeding lines as well as for S allele identification in cruciferous vegetables. F₁ hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F₁ seeds.     

连接介导PCR及其在体内足迹研究中的应用

连接介导聚合酶链反应是以连接反应为基础的单侧PCR技术,首先在DNA片段的一端连接上一个公共连接子,而后在这个连接子与另一个DNA序列特异的引物间扩增.Vent聚合酶的使用,延伸产物捕获及连接子标记选择策略的采用,大大提高了连接介导聚合酶链反应的敏感性.这一技术的发明大大促进了体内足迹等研究的进行.     

Characterization and genetic distance analysis of isolates of Peronosclerospora sorghi using AFLP fingerprinting

Sorghum downy mildew, caused by the obligate oomycete Peronosclerospora sorghi, has been controlled through the use of resistant cultivars and seed treatment with metalaxyl. A recent outbreak in fields planted with treated seed revealed the presence of a metalaxyl-resistant variant. Here, PCR-based methods including amplification from RAPD primers and two systems of automated AFLP analysis have been used to detect DNA-level genetic variation among 14 isolates including metalaxyl-resistant and susceptible isolates, as well as representatives of common pathotypes 1 and 3 and a new pathotype. In total, 1708 bands were detected after amplification of EcoRI/MseI fragments with 16 primer combinations. Nearly as many amplified products were observed using eight primer pairs with three-base extensions (LI-COR) as with two-base extensions (ABI-Prism genetic capillary system). Approximately 25 % of the bands were polymorphic across the 14 isolates, with the majority of differences specific to the pathotype P1 isolate. The AFLP banding patterns are consistent with metalaxyl resistance and the new pathotype having evolved from pathotype 3.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Amplification and detection of polymorphic sequence-tagged sites in<Emphasis Type="Italic">Lathyrus sativus</Emphasis>

A simple procedure was developed to convertLathyrus sativus defence-related expressed sequence tags (ESTs) into mappable genetic markers by using PCR. Twenty-nine STS primer pairs were generated on the basis of sequence information from anL. sativus cDNA library. These primers were used to screen for polymorphisms between 2L. sativus accessions, ATC 80878 and ATC 80407, resistant and susceptible, respectively, toMycosphaerella pinodes infection. All 29 primer pairs amplified PCR products in both accessions, 11 of which amplified multiple RAPD-like products. The remaining 18 primer pairs amplified single monomorphic products. Following cloning, sequencing, and database searches, 17 of 18 PCR products were confirmed to have amplified the targeted genome region. Ten of these 17 STS primer pairs revealed polymorphisms between ATC 80878 and ATC 80407 when PCR products were digested with a range of restriction endonucleases. These results suggest that the STS-based PCR analysis will be useful for generating informative molecular markers inL. sativus for future genome mapping experiments.     

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique. The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments. Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant, indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied.     

Glutamine Synthetase Activity, Relative Water Content and Water Potential in Maize Submitted to Drought

Primer screening and optimization for random amplified polymorphic DNA (RAPD) analysis of cashew (Anacardium occidentale L.) was investigated. Among four series (A, B, D and N) of 10-mer primers, A-series performed better amplification of fragments than other series. The maximum amplification fragments was obtained using OPA-02, OPA-03, OPA-09, OPB-06, OPB-10, OPD-03, OPD-05 and OPN-03 primers. The primers OPA-02 and OPN-03 produced maximum number of DNA fragments in Anacardium occidentale cv. H-320. Primers (OPB-08 and OPN-05 performed a least number of amplification fragments. RAPD profile also indicate that some primer did not produce good amplification. The primer OPA-02 amplified 12 number of polymorphic bands in 20 cultivars of cashew. Only one DNA fragment was produced in A. occidentale cv. Vridhachalam - 2 (M-44/3) by using the primer OPA-02. This revised version was published online in July 2006 with corrections to the Cover Date.