辽宁绒山羊CSN3基因PCR-RFLP多态性分析

本试验以辽宁绒山羊和内蒙古绒山羊为实验动物,采用PCR-RFLP技术对-酪蛋白基因(CSN3)片段的Ⅰ的酶切多态性进行分析,结果表明:辽宁绒山羊和内蒙古绒山羊2个山羊群体中均存在CSN3基因Ⅰ酶切位点的多态性,且均有A、B两个等位基因。辽宁绒山羊群体等位基因A和B的基因频率分别为0.46和0.54;内蒙古绒山羊群体中等位基因A和B的基因频率分别为0.31和0.69。CSN3基因Ⅰ酶切位点的基因型分布在辽宁绒山羊群体中极显著偏离Hardy-Weinberg平衡定理(<0.01),在内蒙古绒山羊群体中符合Hardy-Weinberg平衡定理(>0.05),但两个群体间存在显著差异(<0.01)。     

Control and monitoring: monitoring and eradication of sharka in south-east Italy over 15 years

When the first foci of sharka were discovered in Puglia region (south-east Italy) in the late 1980s, the regional agricultural authorities launched a programme for Plum pox virus (PPV) monitoring and disease eradication. The infecting virus strain was identified as PPV-D. From 1989 to 1993, a strong eradication campaign was successfully carried out involving 13 plum and 2 apricot orchards with different levels of infection. During 1994–2000, besides plum, apricot and peach, monitoring was extended to sweet cherry. At that time, surveys and testing did not reveal any new PPV focus, but the eradication of infected trees continued in a couple of orchards. In 2001–05, particular attention was paid to peach, as devastating PPV-M outbreaks had developed in other areas of the country. A new PPV focus was found in apricot, caused by PPV-Rec, which was promptly eradicated. In the following two years, surveys in the once infected orchard and surrounding peach plantings did not detect any virus spread. The endeavour has taken 15 years making this PPV monitoring and eradication programme the longest in Italy. Its overall results indicate that the fruit tree industry in Puglia region can now be regarded as essentially PPV-free.     

Effects of three esca-associated fungi on Vitis vinifera L.: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines

Secondary metabolites and host defense compounds were shown to occur in xylem sap, and leaves of Vitis vinifera cv. Italia and cv. Matilde naturally infected by the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme). Samples of xylem sap and leaves were collected from healthy vines and from vines showing severe symptoms of brown wood-streaking caused by Pch and Tmi, or from vines with symptoms of both brown wood-streaking and white rot caused by Fme. Xylem sap collection was carried out during the early spring of 2003 and 2004, corresponding to the phenological phases: (A) cotton bud; (B) green tip; (C) leaves out; (D) stretched out leaves; and (E) visible clusters. In the present work we have studied the accumulation of biomolecules (pentaketides and α-glucans), host defense compounds (benzaldehydes, benzoic acid and cinnamic acid derivatives, flavonols, flavanols, flavan-3-ol derivatives and stilbenes) at different stages of grapevine development. Accumulation and changes in total phenolics and recurring phenolics, and of three phytotoxic secondary metabolites (scytalone, isosclerone and pullulan) were analyzed by HPLC. On comparing results for cv. Italia and cv. Matilde, it can be seen that phenolic concentrations are strongly related to the cv.     

Effects of three esca-associated fungi on Vitis vinifera L.: III. Enzymes produced by the pathogens and their role in fungus-to-plant or in fungus-to-fungus interactions

When the esca-associated fungi Phaeomoniella chlamydospora (Pch), Togninia minima (Tmi) and Fomitiporia mediterranea (Fme) were grown in liquid stationary cultures, it was seen that they were able to live in media containing resveratrol (RES) or tannic acid (TA) as the sole carbon source and that the fungi were able to convert both compounds. Particular attention is paid here to detecting RES and TA conversion. Pch, Tmi and Fme were partially inhibited by RES or TA. Pch, Tmi and Fme produced extracellular tannase, laccase and peroxidase enzymes in liquid or agarized cultures, whether glucose was present or not. When colonies of Pch, Tmi and Fme were confronted, they showed spatially and temporally heterogeneous patterns of laccase and peroxidase activity. The results indicate the non-synergistic, competitive association of Pch and Tmi and the inhibition of Fme growth. Muconic acid, a well-known intermediate in a large number of lignin and phenol oxidative processes, can partly or completely inhibit the lignolytic agent Fme, but is tolerated by Pch and Tmi. An explanation for wood pigmentation patterns by Pch, Tmi and Fme is given.     

实时定量PCR技术在瘤胃微生物定量分析中的研究应用

随着对瘤胃微生物研究的逐步深入,一些以分子生物学技术为基础的定量分析方法对瘤胃微生物的研究方兴未艾。本文简要介绍了实时定量PCR技术的原理、操作程序以及该技术在反刍动物瘤胃微生物定量分析中的应用现状、取得的成果和前景展望。     

猪链球菌对红霉素耐药性的研究

从发病猪体内分离、鉴定猪链球菌，采用肉汤稀释法和纸片琼脂扩散法筛选对红霉素耐药的猪链球菌，用双纸片法确定耐药株的耐药表型，通过聚合酶链反应检测对红霉素耐药的基因ermb/mefA。猪链球菌对红霉素的耐药表型为cMLS表型，即同时对克林霉素耐药。在3株红霉素耐药株中扩增到ermb基因，其余未能检测到ermb或mefA基因。     

Validation of RT‐PCR Assays for Molecular Characterization of Porcine Teschoviruses and Enteroviruses

Porcine enteroviruses (PEVs) and teschoviruses (PTVs) are described as causative agents of neurological disorders, fertility disorders and dermal lesions of swine. Difficulties in the serological detection of these viruses may lead to a significant underestimation of infections with clinical symptoms. With the recent availability of genome sequence data for all the serotypes, molecular diagnosis is a possibility. The present study describes a new approach to molecular ‘serotyping’ of PTVs and PEV‐B viruses, involving the amplification and sequencing of a genomic fragment of the VP1 coding region. A molecular characterization of Italian entero‐teschovirus isolates was performed using a set of previously published and newly designed polymerase chain reaction primers. A total of 33 porcine isolates and 10 reference strains were analysed. Porcine enterovirus‐B samples were first diagnosed as positive for enterovirus by amplification of the 5′‐non‐translated region. Samples were then typed by amplification and sequencing of a portion of the VP1 coding region. Porcine enterovirus‐A and PTVs were detected by a published assay in the 5′‐NC region that allows them to be differentiated according to the size of amplification product, using the same set of primers. For serotype characterization of PTV, we evaluated four different regions: the N terminus of the capsid protein VP2, the region encoding for RNA‐dependent RNA polymerase, and the capsid VP1 and VP4 regions. The newly designed primers in the VP1 region was proved to be broad in range and suitable for serotype assessment and therefore constitute a useful diagnostic tool for molecular diagnosis of porcine teschovirus/enterovirus strains and for the study of molecular epidemiology and evolution of these viruses.     

Synthesis and insecticidal activities of pyridine ring derivatives of podophyllotoxin

In an attempt to find the biorational pesticides, we synthesized 12 pyridinyl derivatives of podophyllotoxin (PPT) and 4′-demthylepipodophylltoxin (4′-DMEP) in this study. Their structures and the α/β substitution at C-4 were confirmed by ¹H NMR, IR, MS spectral analyses and elemental analysis. The insecticidal activities were tested against fifth-instar larvae of Pieris rapae and the third-instar larvae of Cullex pipiens pallens at concentrations of 250 and 10 μg ml⁻¹. Four derivatives of PPT, 4.1, 4.2, 4.3 and 4.5, showed higher insecticidal activities against P. rapae than PPT, while three derivatives of PPT, 4.4, 4.5 and 4.6, displayed higher mosquito larvicidal activity than PPT, with LC₅₀ values of 1.66, 3.96 and 1.54 mg l⁻¹, respectively. Interestingly, we also found that the pyridine ring derivatives of PPT showed delayed insecticidal activity, which is different from traditional neurotic insecticides. The results suggest that 4′-OCH₃ in the PPT derivatives is essential to keep the insecticidal activity and the insecticidal activities of pyridine ring derivatives of PPT are higher than that of the derivatives of 4′-DMEP, supporting PPT has the potential to be a lead structure of semi-synthetic insecticides.     

表达猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白重组鸡痘病毒的构建

从含有猪繁殖与呼吸综合征病毒（PRRSV）核衣壳蛋白（N）的质粒扩增出N基因，构建禽痘病毒转移载体。该载体含有禽痘病毒早晚期启动子LP2EP2控制之下的PRRSVN基因、P11启动下的报告基因lacZ以及用于同源重组的禽痘病毒基因组的片段。在转移载体转染亲本病毒S—FPV-017感染的鸡胚成纤维细胞（CEF）之后，采用蓝色表型筛选的方法，筛选到表达N基因的重组病毒，并对其进行了6轮蚀斑纯化。PCR方法鉴定证明重组病毒的基因组中含有完整PRRSVN基因，间接免疫荧光试验证明了PRRSVN蛋白在重组病毒感染的CEF细胞中获得表达，本研究为猪繁殖与呼吸综合征非复制型疫苗的研制打下了基础。