牛支原体氟喹诺酮类药物耐药靶位突变分析

为探讨牛支原体氟喹诺酮类药物耐药靶位的突变情况,对分离自我国多个省份的牛支原体进行氟喹诺酮类药物耐药性检测和耐药株筛选,通过对临床分离敏感株、耐药株及体外诱导高度耐药株的氟喹诺酮类药物耐药决定区(QRDR)进行测序分析,发现分离菌株中有19%(6/32株)对氟喹诺酮类药物耐药,其QRDR中均存在gyr A(Ser83Phe)或par C(Ser80Ile)的氨基酸突变;但在体外诱导的高度耐药株中QRDR突变类型则以gyr A(Ser83Phe/Tyr或Glu87Lys)和par C(Ser80Ile或Asp84Asn/Tyr)的氨基酸发生突变为主,以上靶位突变在介导牛支原体对氟喹诺酮类药物耐药水平方面是否起着决定性作用,还有待进一步证明。     

柱花草炭疽菌致病力丧失突变菌株1869的T-DNA插入位点侧翼序列的克隆

通过对实验室已构建的柱花草炭疽菌T-DNA突变体库中各转化子致病力的测定,获得致病力丧失突变菌株1869。对其进行PCR检测以验证T-DNA在1869基因组中插入情况,并测定观察其菌落直径、菌落形态及产孢量等生物学特性,利用TAIL-PCR克隆标记基因侧翼序列,采用生物信息学方法比对基因信息。结果表明,与柱花草炭疽菌野生型菌株CH008相比,突变菌株1869表现致病力丧失,菌落生长速率也显著低于野生型;然而,在分生孢子形态、产孢能力、孢子萌发率等方面与野生型并无明显差异。TAIL-PCR扩增得到T-DNA插入位点RB端序列467bp,LB端侧翼序列388bp,经两侧序列拼接比对,所得序列与Pac1同源性达到92%~96%,推测可能由于基因表达产物调节菌株对pH值的敏感性,从而影响了其致病过程。     

Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test

Populations of the codling moth, Cydia pomonella L (Lepidoptera, Tortricidae) have developed resistance to several classes of insecticide such as benzoylureas, juvenile hormone analogues, ecdysone agonists and pyrethroids, but the corresponding resistance mechanisms have not been extensively studied. Knockdown resistance (kdr) to pyrethroid insecticides has been associated with point mutations in the para sodium channel gene in a great variety of insect pest species. We have studied two susceptible strains (S and Sv) and two resistant strains (Rt and Rv) of C pomonella that exhibited 4- and 80-fold resistance ratios to deltamethrin, respectively. The region of the voltage-dependent sodium channel gene which includes the position where kdr and super-kdr mutations have been found in Musca domestica L was amplified. The kdr mutation, a leucine-to-phenylalanine replacement at position 1014, was found only in the Rv strain. In contrast, the super-kdr mutation, a methionine-to-threonine replacement at position 918, was not detected in any C pomonella strain. These data allowed us to develop a PCR-based diagnostic test (PASA) to monitor the frequency of the kdr mutation in natural populations of C pomonella in order to define appropriate insecticide treatments in orchards.     

A psbA mutation in Kochia scoparia (L) Schrad from railroad rights-of-way with resistance to diuron, tebuthiuron and metribuzin

Kochia [Kochia scoparia (L) Schrad] has become resistant to many herbicides used in cropland and railroad rights-of-way in North Dakota and Minnesota. Kochia scoparia plants that had survived annual treatments with diuron and tebuthiuron were sampled along railroad rights-of-way in North Dakota and Minnesota. The samples were screened in the greenhouse for resistance to diuron, tebuthiuron, metribuzin and bromoxynil from 0.5x to 32x the recommended use rates. A resistant K scoparia accession (MN-3R) was confirmed with resistance up to 16-fold higher than recommended use rates for tebuthiuron and diuron and up to 4-fold higher for metribuzin. However, the resistant K scoparia accession was susceptible to bromoxynil even at 50% of the recommended use rate. The herbicide binding region of the psbA gene fragment of eight resistant (R) and seven susceptible (S) K scoparia accessions was PCR-amplified and sequenced for detection of mutations. The psbA gene of four R K scoparia accessions was mutated at residue 219 with substitution of isoleucine for valine (GenBank accession number AY251265). The seven S K scoparia accession sequences were wild-type at this residue (GenBank accession number AY251266). The other four R accessions sequences showed a previously known triazine R mutation with substitution of glycine for serine at residue 264. All 15 K scoparia accessions were wild-type at all other psbA residues within the region analyzed. Resistance to diuron, tebuthiuron and metribuzin among the railroad rights-of-way K scoparia is probably due to the mutation at residue 219 of the psbA gene in some plants, but due to the previously reported Ser(264)Gly substitution in other plants. Target-site resistance associated with a change of valine to isoleucine at residue 219 of the psbA target-site in weeds has previously been reported for Poa annua L selected in diuron-treated grass seed fields, and for Amaranthus powelli S Wats selected in linuron-treated carrot fields. This is the first report of the mutation in herbicide-resistant K scoparia.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation as a tool for random mutagenesis of <Emphasis Type="Italic">Colletotrichum trifolii</Emphasis>

We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene (hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into the fungal genome. More significantly, about 75% of transformants had a single copy of the T-DNA. The results demonstrate that insertional mutagenesis via A. tumefaciens is a useful tool for studying the function of C. trifolii genes.     

Monitoring and characterization of Magnaporthe grisea isolates with decreased sensitivity to scytalone dehydratase inhibitors

Rice blast fungus isolates were collected in Kyushu to investigate resistance to scytalone dehydratase inhibitors of melanin biosynthesis (MBI-D). In 2001, failure of control of rice blast was reported in the Saga prefecture, where MBI-Ds have been used since 1998. At that time, the distribution of resistant isolates was mainly limited to that area. However, in 2002, resistant isolates were detected in all prefectures of Kyushu. DNA fingerprinting analysis showed that the mutation causing resistance to MBI-Ds had arisen independently in each area. These data suggest that resistant isolates may occur in any area and become dominant under continuous selection pressure for MBI-Ds. Nevertheless, resistant strains can be controlled by reductase inhibitors of melanin biosynthesis (MBI-R) or commercial rice seed disinfectants.     

猪繁殖与呼吸综合症病毒生物学特性

猪繁殖与呼吸综合征(PRRS)为当前危害世界养猪业的重要疾病之一.本文根据当前各国学者对此病原的研究成果从病毒的分类地位,形态、理化特性,组织分布,培养特性,免疫学特性,病毒的基因组成、表达及其功能,病毒蛋白及功能,基因组的遗传和变异作一个系统的综述.     

Gain‐of‐function mutation in PTPN11 in histiocytic sarcomas of Bernese Mountain Dogs

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm of dendritic cell origin that is common in certain breeds of dogs. High prevalence of fatal, disseminated HS has been described in Bernese Mountain Dogs (BMDs). Support for genetic predisposition to develop HS has been presented in several studies, but to date, causative genetic events have not been reported. In addition, no driver mutations have been identified in tumours. Recently, E76K gain‐of‐function mutation in SHP2 encoded by the PTPN11 gene has been described in human histiocytic malignancies. In our study, we identified the PTPN11^E76K in HS of BMDs. Amplification of exon 3 of the PTPN11 gene followed by Sanger sequencing was used to detect the mutation and estimate the prevalence in HS from 30 BMDs, 13 Golden Retrievers and 10 other dog breeds. The overall prevalence of PTPN11^E76K in HS of BMDs was 36.67% compared with 8.69% in other breeds. No mutation was identified in normal tissues from 10 BMDs with HS that carried the mutation and 12 control dogs with no neoplastic disease, including 6 BMDs. Increased immunoreactivity for AKT, phosphorylated ERK1/2 and phosphorylated AKT in a small subset of BMDs with PTPN11^E76K suggests that a gain‐of‐function might be mediated by the ERK and AKT pathways. These data suggest PTPN11^E76K as an important driver mutation of HS in BMDs. This information may not only aid in unravelling the tumourigenic events associated with HS in BMDs, but also help in identifying more promising therapeutic strategies.