盐生植物小花碱茅外整流K┼通道SKOR基因片段的克隆及序列分析

为研究小花碱茅(Puccinellia tenuiflora)K+/Na+选择性运输的分子机制，根据已知的外整流K+通道高度保守区设计一对简并性引物，以小花碱茅根总RNA为模板，采用RT PCR方法克隆SKOR基因片段。序列分析结果表明,该基因片段长度为555 bp，编码185个氨基酸；该序列与其他已报道的高等植物SKOR核苷酸序列的同源性在66%以上，氨基酸序列同源性在55%以上。     

盐生植物小花碱茅K+通道PtAKT1基因片段的克隆及序列分析

为研究小花碱茅(Puainellia tenuiflora)K⁺/Na⁺选择吸收分子机制,根据已报道的其他植物AKT1基因的保守序列设计一对简并性引物,以小花碱茅根部总RNA为模板,采用RT-PCR方法克隆出AKT1基因,以期为小花碱茅AKT1全长基因的克隆、表达调控及其作物改良的研究奠定基础。序列分析结果表明:该基因长度大约为1019 bp,编码339个氨基酸;所得序列与GenBank中注册的高等植物AKT1基因序列的同源性均在85%以上,与其他AKT1的氨基酸序列同源性达73%以上。     

盐生植物盐地碱蓬质膜Na^＋/H^＋逆向转运蛋白基因片段的克隆及其序列分析

为研究盐地碱蓬(Suaeda salsa)Na+长距离运输的分子机制,根据其他植物质膜Na+/H+逆向转运蛋白SOS1基因的保守序列设计一对简并引物,以盐地碱蓬根系总RNA为模板,采用RT PCR方法克隆出SOS1基因片段,以期为盐地碱蓬SOS1全长基因的克隆、表达调控等研究奠定基础。序列分析结果表明,该基因片段长度为736 bp,编码244个氨基酸;该序列与其他高等植物SOS1核苷酸序列的同源性在74%以上,氨基酸序列同源性则在61%以上。     

多浆旱生植物霸王液泡膜H+-PPase基因片段的克隆及序列分析

根据其他植物H⁺-PPase基因的保守序列设计一对简并性引物,以霸王叶片总RNA为模板,采用RT-PCR的方法扩增出H⁺-PPase基因片段并克隆到PUCm-T载体。阳性克隆经PCR鉴定后进行测序,序列分析结果表明,克隆到3个同源的序列片段(898 bp),编码299个氨基酸;所得序列与GenBank中注册的高等植物H⁺-PPase基因核苷酸序列的同源性均在69%以上、氨基酸序列的同源性达78%以上。     

微孔草Actin基因核心片段的克隆及序列分析

提取微孔草Microula sikkimensis叶片的总RNA,并以其为模板,运用RT-PCR方法扩增出Actin基因的核心序列。通过连接、转化后对阳性克隆进行PCR鉴定并测序,序列分析结果表明:微孔草Actin基因核心片段长599 bp,编码199个氨基酸。将该序列与GenBank中已注册的植物Actin基因序列进行同源性比较,发现它们之间的同源性在84%以上,氨基酸序列同源性在95%以上,具有高度的保守性。     

小花碱茅HKT2;1基因全长cDNA的克隆与生物信息学分析

Na⁺是盐渍化土壤中主要的毒害离子,对植物生长发育和农业生产构成严重威胁。高亲和性K⁺转运蛋白HKT2;1在控制高等植物Na⁺吸收,增强K⁺的选择性,进而提高耐盐性方面发挥着重要作用。本研究以拒盐型牧草小花碱茅为材料,采用RT-PCR和RACE(rapid amplification of cDNA ends)方法克隆到HKT2;1基因,并命名为PutHKT2;1。该基因全长1 919 bp,包含1个长1 638 bp的开放阅读框(ORF),编码546个氨基酸,推测分子量为60.5 kDa,等电点PI为9.07。与其他植物HKT2;1氨基酸序列同源性多在66%以上,核苷酸序列同源性都在75%以上。PutHKT2;1可能跨膜11次,二级结构分析表明,PutHKT2;1蛋白含有47.99% α-螺旋、5.13% β-转角、31.87%无规则卷曲和15.01%延伸链。PutHKT2;1基因全长cDNA的克隆及其生物信息学分析为进一步揭示小花碱茅拒盐的分子机制奠定了基础。     

黑曲霉3928植酸酶phy基因的克隆及全序列分析

根据GenBank中已注册的黑曲霉基因序列设计了1对引物,通过PCR方法将黑曲霉phy A全基因进行扩增,获得了1条长约1.5 kb的特异性PCR产物,在PMD18 T载体中克隆了黑曲霉3928植酸酶phy A全基因,筛选到2个重组菌落(1#和2#),并进行了全序列测定。序列分析结果表明:2个重组质粒的测序结果完全一致;克隆的片段中含有完整的phy A基因序列,全长1 506 bp,其中含有一段长102 bp的内含子;phyA基因全序列编码467个氨基酸,信号肽位于基因的5′端,长度为19个氨基酸;碱基序列与GenBank中报道的AY513749的同源性为98.87%,编码的氨基酸序列的同源性为97.21%。     

山羊痘病毒ITR基因片段的克隆与序列分析

应用PCR方法扩增山羊痘病毒QL、LD、Y和B株ITR基因片段,插入pMD18-T载体,转化DH5α感受态细胞,提取重组载体经PCR和酶切鉴定后测序,并与GenBank上8株羊痘病毒相应序列进行同源性分析。结果经PCR扩增,4株病毒均可得到一条均一的DNA片段,该片段可被内切酶AluⅠ特异酶切,而且该PCR的DNA模板最小检出量为24.40pg。经测序,该基因片段长度为289bp;序列比较发现,4株病毒ITR片段与GenBank上2株山羊痘病毒的核苷酸与氨基酸序列同源性均为100%,与3株绵羊痘病毒分别为98.2%和96.5%,与3株牛疙瘩皮肤病病毒为98.2%～99.3%和96.5%～97.7%。这表明ITR基因片段在羊痘病毒属中较为保守,可以针对ITR基因建立羊痘病毒的PCR检测方法。     

猪巨细胞病毒(PCMV)四川株gB基因的克隆与序列分析

根据GenBank中猪巨细胞病毒(porcine cytomegalovirus,PCMV)gB基因的核苷酸序列(GenBank登录号:FJ870563.1)设计1对引物,采用PCR方法从确诊为猪巨细胞病毒的阳性样品中扩增gB基因,将其克隆到pMD19-T载体上,转化DH5α感受态细胞,提取重组质粒T-PCMV-gB,经PCR和酶切鉴定后测序,并与GenBank上gB相应序列进行同源性分析。结果表明,该基因片段长度为2580 bp,与其他参考株gB基因的核苷酸同源性达98.0%～99.6%;其推导的氨基酸序列与人疱疹病毒6型和7型比较分析结果显示,其同源性分别为42.9%～43.6%和40.5%～40.6%。这些氨基酸差异对病毒生物学特性的影响有待于进一步研究。     

丝状支原体丝状亚种SC型中国不同地区分离株脂蛋白Q基因的分子特征

根据已发表的丝状支原体丝状亚种SC型（MmmSC）脂蛋白Q（LppQ）基因序列设计并合成一对引物，利用PCR扩增方法，从我国不同省区的MmmSC分离株HVRI—X、BenI、QingI、MuI和模式株PG1中获得了LppQ基因1303 bp的片段，将该片段克隆到PMD18-T载体上，通过对所得到的重组质粒进行酶切分析、PCR鉴定，证明得到了含有目的基因片段的阳性重组质粒。采用Sanger’s双脱氧末端终止法对插入片段进行核苷酸序列测定，获得了HVRI-X、Ben Ⅰ、oing Ⅰ、Mu Ⅰ株和模式株PG1 LppQ基因核苷酸序列。核苷酸序列比较结果显示，国内4个MmmSC分离株与GenBank公布的LppQ基因核苷酸序列同源性均在99．7％以上，氨基酸序列同源性均在99％以上；与模式株PG1的核苷酸序列同源性在99．7％以上，氨基酸序列同源性均在99．3％以上。，国内4个MmmSC分离株之间的核苷酸序列同源性均在99．7％以上，氨基酸序列同源性均在99．3％以上。对HVPI X株LppQ基因氨基酸亲私陛和蛋白表面可能性进行了分析，证实LppQN末端富含亲水氨基酸，比C末端更有可能定位在蛋白表面。     

拒盐型牧草小花碱茅PutHKT2；1基因表达模式分析

本研究以拒盐型盐生牧草小花碱茅(Puccinellia tenuiflora)为材料,采用RT PCR方法对小花碱茅PutHKT2;1基因表达模式进行研究,以期阐明其在盐胁迫下拒Na+的分子机制。结果表明,PutHKT2;1基因表达存在组织特异性,主要在根中表达,地上部表达量相对较低;K+饥饿处理条件下,在根中的表达量最高;在1 mmol·L-1 K+或无K+条件下,根中PutHKT2;1表达量随着盐浓度的增大而减少。由此推断,PutHKT2;1蛋白主要在根部发挥作用,主要用于控制Na+吸收,增强K+选择性吸收能力,从而维持地上部较高的K+/Na+,提高其耐盐性。     

长穗偃麦草EeHKT1;4基因的克隆与植物表达载体构建

采用RT-PCR方法从长穗偃麦草(Elytrigia elongata)中克隆得到高亲和K+转运蛋白基因,命名为EeHKT1;4,全长cDNA序列为1 977bp,开放阅读框1 722bp,编码573个氨基酸,与一粒小麦(Triticum monococcum)TmHKT1;4-A2的氨基酸同源性为94%。系统进化树分析结果显示,EeHKT1;4与单子叶植物HKT1;4亲缘关系较近。利用In-Fusion技术,成功构建了pBI121-35SEeHKT1;4-Nos植物表达载体,为长穗偃麦草EeHKT1;4的耐盐功能鉴定奠定基础。     

Comparison of the Polymerase Chain Reaction and Culture for the Detection of Feline Chlamydia psittaci in Untreated and Doxycycline-Treated Experimentally Infected Cats

The diagnostic sensitivity of the polymerase chain reaction (PCR) was compared with that of culture on conjunctival swabs over the course of infection in 4 doxycycline-treated and 4 untreated cats that were experimentally infected with feline Chlamydia psittaci. Treated cats were given 25 mg (5 mg/kg) of doxycycline orally twice daily for 3 weeks from day 6 after challenge. Clinical signs improved within 3 days of institution of treatment. Culture remained positive for 1 day and PCR remained positive for up to 5 days after treatment was commenced. No recurrence of clinical signs occurred and the organism could not be detected by either PCR or culture for 2 weeks after cessation of therapy. In the 4 untreated cats, conjunctival swabs were taken daily to day 14 and every 2nd weekday to day 64 after challenge. PCR was significantly more sensitive than culture in untreated cats overall (PCR 85.7%, culture 72.9%, P approximately 0) and for cats with clinical signs (PCR 89.2%, culture 79.2%, P = .008). PCR and culture had equivalent sensitivity (100%) for cats showing clinical signs in the 1st month of infection, whereas PCR was considerably more sensitive than culture for cats showing clinical signs in the 2nd month (PCR 72.9%, culture 47.9%, P = .028). Organisms were not detected by PCR in blood or any tissue collected from treated or untreated cats at postmortem. Thus, effective treatment of chlamydiosis in cats is possible with much shorter treatment regimens than currently recommended, and PCR is the more sensitive diagnostic method in chronically infected cats.     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.     

HKT蛋白与植物耐盐性研究进展

盐胁迫是一种严重影响植物生长的非生物胁迫,植物在长期进化过程中形成了各种机制来抵御和适应盐胁迫。高亲和性钾离子转运蛋白(HKT)是植物中普遍存在的一类蛋白,对高等植物体内Na+再循环,维持植株地上部特别是叶片中的低Na+浓度和高K+/Na+具有重要作用。本研究对HKT蛋白的分子特性、结构与功能及其与植物耐盐性的关系进行了综述。     

Detection of Porcine circovirus type 2 viremia and seroconversion in naturally infected pigs in a farrow-to-finish barn

Porcine serum was assayed by 2 polymerase chain reaction (PCR) protocols (nested PCR [nPCR] and non-nested PCR) and a competitive enzyme-linked immunosorbent assay to determine when Porcine circovirus type 2 (PCV2) viremia and a rise in the serum level of PCV2-specific antibody occurred in pigs raised in a large Canadian farrow-to-finish barn. Eight serial blood samples were collected from each of 40 pigs from 5 to 156 (+/- 1.5) d of age; 6 pigs were removed from the study for various reasons at various times. Viremia was not detected in the samples collected before 72 d of age but was detected in those collected on or after 72 d: of 33 pigs, 7 (21%) had only 1 serum sample positive for PCV2 DNA by nPCR after day 72; 11 (33%) were intermittently positive by nPCR, non-nested PCR, or both between 72 and 156 d; and the remaining 15 (45%) were repeatedly positive (in 2 to 4 samples). The level of serum antibody against PCV2 declined after weaning and increased between 72 and 107 d of age, only after PCV2 was detected in serum. Our results show that PCV2 viremia persists in the presence of elevated levels of PCV2-specific antibody.     

Molecular diagnosis of alcelaphine herpesvirus (malignant catarrhal fever) infections by nested amplification of viral DNA in bovine blood buffy coat specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.