T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

Feline Ubiquitin Fusion Protein Genes

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Cloning of p15INK4b related gene CCRG and expression analysis in the renal carcinoma

AIM and METHODS:To analysis the factor that involved in renal carcinogenesis, we used the bait gene AK001518 to screen GenBank. To understand the relationship between cell cycle related gene(CCRG) and p15, we did RT-PCR and Northern Blot experiments. Then we examined CCRG expression level in renal carcinogenesis. RESULTS:Gained a function unknown gene CCRG that was 67% a mino acid identical with the gene AK001518 that was regulated by p15. It was shown that the CCRG mRNA was dramatically decreased when p15 gene was over-expressed. CCRG expression level was much higher in tumor tissues and cells than normal tissues and cells. CONCLUSION:The novel gene CCRG expressed highly in the renal carcinoma, which might play a significant role in the renal carcinogenesis.     

Identification of ALS inhibitor‐resistant Amaranthus biotypes using polymerase chain reaction amplification of specific alleles

Summary Polymerase chain reaction (PCR) amplification of specific alleles (PASA) was adapted as a molecular marker‐based method for the rapid detection of point mutations in Amaranthus retroflexus and Amaranthus rudis leading to ALS inhibitor resistance. Two pairs of primers were designed for the specific amplification of alleles of the ALS gene of susceptible and resistant biotypes. The allele‐specific primer matched the desired allele, but mismatched the different allele at its 3′ end. Differentiation was carried out by comparison of the amplified DNA fragments in gel electrophoresis after PASA‐PCR. In A. rudis, differentiation was possible with one PCR and genomic DNA as probe. A ‘nested’ PCR was necessary for the differentiation of sensitive and resistant A. retroflexus. PASA is useful for the identification of resistant weed biotypes and also as a monitoring tool to map resistance occurrence and distribution. Advantages include the fast and clear separation of those plants with and without mutations at an early stage of development, its easy and consistent performance and quick results compared with existing resistance detection tests. These advantages, when combined with management strategies, enable further activities to reduce herbicide resistance.     

Role of polymerase β in DNA metabolism and genomic instability

The major role of DNA polymerase β was thought to be limited in its involvement in short patch base excision repair by removing 5’-deoxyribose phosphate and base insertion. However, the recent researches indicate that polymerase β might take part in a wide spectrum of DNA metabolism reactions, including long patch base excision repair, DNA replication, recombination, meiosis and transleisional DNA synthesis. Because of its wide and important cellular function, an inappropriate intracellular polymerase β level might be associated with genomic instability. Down-regulation or mutation of polymerase β is mutagenic due to deficient in DNA repair, while overexpression of this error-prone β polymerase might perturb the normal function of other accurate polymerases and cause genomic instability as well.     

Seasonal distribution of phytoplasmas in Australian grapevines

The distribution and persistence of phytoplasmas were determined in Australian grapevines. Phytoplasmas could be detected using the polymerase chain reaction (PCR) from shoots, cordons, trunks and roots throughout the year, and phytoplasmas appear to persistently infect Australian grapevines from year to year. Phytoplasmas were not always detected in samples from the same sampling area from one sampling period to the next. Phytoplasma detection by PCR was improved by sampling from shoots, cordons and trunks, especially during October (early spring). The diseases expressed by the 20 grapevines used in the distribution and persistence studies were monitored. Australian grapevine yellows disease (AGY) was expressed by 17/20 grapevines at some time during the study, whilst only 4/20 and 15/20 grapevines expressed restricted growth disease (RG) and late season leaf curl disease (LSLC), respectively. All grapevines with RG and LSLC also had AGY. The three diseases were persistently expressed in some grapevines and remission of disease was observed in others. The results of PCR detection in the same grapevines indicated that phytoplasmas were more frequently detected in AGY-affected grapevines that also expressed RG and LSLC compared with grapevines expressing AGY alone. Phytoplasmas were detected in symptomless plant material but less frequently compared with AGY-affected material.     

刺梨及其近缘种PCR实验体系的建立与优化

以刺梨及其近缘种月季为试材,进行了RAPD-PCR实验参数的确立和优化试验。结果表明,刺梨及月季25μL反应体系的最优组成为2.5μL10×反应缓冲液,2mmol/LMg2+,0.2mmol/LdNTP,1.6mg/L模板DNA,0.4μmol/L随机引物和1.2UTaqDNA多聚酶。经PCR扩增验证,此反应体系亦适宜于刺梨的部分近缘种,可有效用于RAPD分析;通过将退火温度提高至50℃或采用“Touchdown”扩增程序,并在50μL反应体系中适当增加特异引物对浓度(1.0μmol/L),模板DNA(4.0mg/L)和TaqDNA多聚酶(3.0U/管)的使用量,建立起适合于刺梨特异DNA片段检测及回收的特异PCR扩增实验体系,为刺梨的分子克隆奠定了技术基础。     

Mariner转座子与小菜蛾抗性关系的研究

以抗溴氰菊酯、杀虫双、杀螟丹小菜蛾种群及其敏感品系为研究对象,借助聚合酶链式反应(PCR)、载体筛选、琼脂糖凝胶电泳等实验技术,检测了小菜蛾4个品系的Mariner转座子的存在及其与抗性的关系。结果表明,在抗溴氰菊酯、杀虫双、杀螟丹以及敏感品系的小菜蛾中都存在两种大约500 bp的Mariner转座子片断,初次证明在小菜蛾4个品系中均含有两种Mariner转座子,并发现一种新的转座子基因片断,但是没有发现Mariner转座子与抗性之间存在直接的联系。研究结果将为利用Mariner转座子标签法在小菜蛾中分离定位基因、转化外源基因、转基因昆虫防治害虫等研究提供理论基础。     

Trichoderma Biocontrol of Colletotrichum acutatum and Botrytis cinerea and Survival in Strawberry

Trichoderma isolates are known for their ability to control plant pathogens. It has been shown that various isolates of Trichoderma, including T. harzianum isolate T-39 from the commercial biological control product TRICHODEX, were effective in controlling anthracnose (Colletotrichum acutatum) and grey mould (Botrytis cinerea) in strawberry, under controlled and greenhouse conditions. Three selected Trichoderma strains, namely T-39, T-161 and T-166, were evaluated in large-scale experiments using different timing application and dosage rates for reduction of strawberry anthracnose and grey mould. All possible combinations of single, double or triple mixtures of Trichoderma strains, applied at 0.4% and 0.8% concentrations, and at 7 or 10 day intervals, resulted in reduction of anthracnose severity; the higher concentration (0.8%) was superior in control whether used with single isolates or as a result of combined application of two isolates, each at 0.4%. Only a few treatments resulted in significant control of grey mould. Isolates T-39 applied at 0.4% at 2 day intervals, T-166 at 0.4%, or T-161 combined with T-39 at 0.4% were as effective as the chemical fungicide fenhexamide. The survival dynamics of populations of the Trichoderma isolates (T-39, T-105, T-161 and T-166) applied separately was determined by dilution plating and isolates in the mixtures calculated according to the polymerase chain reaction (PCR) using repeat motif primers. The biocontrol isolates were identified to the respective species T. harzianum (T-39), T. hamatum (T-105), T. atroviride (T-161) and T. longibrachiatum (T-166), according to internal transcribed spacer sequence analysis.