半巢式反转录实时荧光PCR检测马铃薯黑环斑病毒

本研究根据基因组中RNA依赖的RNA聚合酶(RDRP)保守序列,设计合成了3条巢式PCR引物和1条TaqMAN荧光探针,建立了半巢式反转录实时荧光PCR检测PBRSV的新方法.该方法采用E.Z.N.A TM快速提取植物总RNA,并有机地结合了巢式PCR和TaqMAN探针检测技术.第二步半巢式反转录实时荧光PCR既是对第一步信号的进一步放大,也是对第一步PCR产物的确认,因此,检测的准确性、灵敏度比巢式PCR、单重Realtime PCR等方法高.实验结果表明,该方法检测灵敏度可达300fg/μL植物总RNA.     

沙拉沙星单克隆抗体制备及鉴定

为制备敏感性好、亲和力高、特异性强的抗沙拉沙星(Sarafloxacin,SARA)单克隆抗体,采用碳二亚胺法合成SARA人工抗原,通过免疫BALB/c小鼠,选择血清效价高且敏感性好的小鼠,采用杂交瘤细胞技术进行细胞融合,筛选分泌抗SARA单克隆抗体(Monoclonal antibody,mAb)的杂交瘤细胞株;采用体内诱生腹水法制备SARA mAb,通过间接ELISA和间接竞争ELISA对SARA mAb的免疫学特性进行鉴定。结果表明:小鼠经免疫原免疫后,小鼠多抗血清效价均达到1∶128 000。选择敏感性较好的2号小鼠进行细胞融合,经多次亚克隆后,筛选出1G3、3H3两株杂交瘤细胞株,其中1G3所产SARA mAb效价较高,为1∶512 000,IC_(50)为6.34 ng/mL,亲和常数为8.55×10~8L/mol,与诺氟沙星的交叉反应率为1.06%,与其他药物交叉反应率均低于0.50%。     

建兰花叶病毒和齿兰环斑病毒的检测技术及脱毒方法研究进展

为了弄清目前建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)病毒检测和脱毒的研究概况,本研究详细阐述了2种病毒的检测方法、采用的脱毒技术和脱毒效率,分析比较了不同技术存在的弊端,指出现有的脱毒方法已不能满足兰科植物脱毒研究的需要。在此基础上,进一步提出应采用安全、可靠的新型脱毒技术来解决目前兰科植物产业瓶颈问题。超低温脱毒技术是近年来发展迅速的脱毒技术,具有独特优势,目前已在多种植物上成功应用,该方法的应用有望解决2种病毒脱毒方面存在的问题。     

朱顶红褪绿环斑病毒侵染辣椒幼苗的miRNA差异表达分析

为分析朱顶红褪绿环斑病毒(Hippeastrum chlorotic ringspot virus,HCRV)侵染的辣椒幼苗与对照健康辣椒幼苗的miRNA差异表达数据,以探索miRNA在HCRV与寄主互作中的机制.本研究采用人工摩擦接种方法接种病毒,1 dpi采样,提取总RNA,利用高通量测序技术检测HCRV侵染样品和...     

抗旋毛虫单克隆抗体杂交瘤细胞系的建立

用旋毛虫感染性的幼虫和排泄分泌抗原免疫BALB/C纯系小鼠,取其脾细胞和SP_2/O—Ag14骨髓瘤细胞,在聚乙二醇(PEG)作用下融合,用间接酶联免疫吸附试验(ELISA)检测,阳性孔用有限稀释法经3～4次克隆化筛选培养,获得了D_4G_6和E_2B_(10)二个杂交瘤细胞系.经3～6个月的传代培养、冻存和接种小鼠诱生腹水,杂交瘤细胞生长良好,保持了稳定分泌抗体的能力.鼠性腹水和血清效价分别为6×10~(-3)和1×10~(-4).抗体免疫球蛋白用免疫琼脂双扩散试验测定均为IgG_1亚类.     

抗猪瘟病毒单克隆抗体的制备及其生物学特性鉴定

为对猪瘟病毒(CSFV)建立更加有效的临床检测方法.用纯化的猪瘟兔化弱毒免疫BALB/c小鼠,取其脾细胞与Sp2/O骨髓瘤细胞融合.经ELISA方法筛选和3次亚克隆,最终获得了5株能稳定传代并分泌抗猪瘟病毒单克隆抗体的杂交瘤细胞株,分别命名为:C7,C9,G9,G10和4E8,其分泌的单克隆抗体(McAb)为IgG1(G9,4E8)和IgG2a(C7.C9,G10)亚类.经鉴定,5株单抗细胞培养上清效价为1:1 600～1:3200,腹水效价为1:51200～1:102400.交叉试验及特异性抗原阻断试验表明,所制备的McAb与其他抗原无交叉反应性,均完全针对猪瘟病毒(CSFV)抗原决定簇,具有高度特异性.稳定性试验表明,制备的杂交瘤细胞株经连续传代25代和经3次冻存复苏后,仍能稳定分泌特异性抗体.抗CSFV McAb的成功制备,为进一步研究CSFV的生物学特性及其快速诊断方法的建立奠定了基础.     

克伦特罗单克隆抗体的制备及其初步鉴定

【研究目的】制备克伦特罗单克隆抗体（McAb）,为建立克伦特罗残留检测方法奠定基础。【方法】用偶氮化法将克伦特罗与钥孔血蓝蛋白（KLH）、牛血清白蛋白（BSA）及卵清白蛋白（OVA）偶联,制备完全抗原,免疫Balb/c小鼠,建立杂交瘤细胞株以制备单抗,并对单抗的特异性、交叉反应性及抗原识别位点等特性进行了初步鉴定。【结果】获得了1H5、1H7、1D6、2F10四株杂交瘤腹水效价均在1：105以上。对沙丁胺醇交叉反应性测定,最小的是1D6,只有2.32%的交叉反应性。单抗相对亲和力1D6〉1H5〉1H7〉2F10。抗原识别位点分析结果表明,这四株单抗至少识别两个不同的抗原位点。选用1D6初步建立了竞争ELISA检测方法,检测限可达1ng/ml,为进一步研制残留检测试剂盒奠定了基础。     

Inheritance and linkage analysis of resistance to zucchini yellow mosaic virus, watermelon mosaic virus, papaya ringspot virus and powdery mildew in melon

A melon (Cucumis melo L.) breeding line derived from PI 414723 is resistant to three potyviruses,watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), papaya ringspot virus (PRSV), and to powdery mildew (PM). The inheritance and linkage relationships of these four resistances were studied in a segregating F2 population and derived F3 families from a cross between cultivar Top Mark and the resistant breeding line. Dominant monogenic inheritance of all four resistances was observed. We report that line 414723-4S3, which was initially selected as a source of ZYMV and WMV resistance, is also a source of dominant monogenic resistances to PRSV and PM race 1. We also report on genetic linkage (significant departure from independent segregation, χ² = 58.1, p≪ 0.0001) between resistance to WMV and ZYMV. The map distance between these loci was estimated to be 7.5 cm. The genes for resistance to PM and PRSV segregated independently from each other, and from ZYMV and WMV resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

圣女果酵素的制备工艺及抗氧化性质研究

以圣女果为原料,以SOD酶活及总酸为检测指标,用液态分步发酵法制备圣女果酵素。优化分步发酵的工艺条件,并对圣女果酵素的抗氧化性质进行研究。结果表明,水添加量50%,白砂糖添加量20%,发酵温度32℃,活性干酵母接种量0.5%,pH值4.1,发酵时间16 h进行一次发酵;发酵温度30℃,乳酸菌接种量0.1%,pH值5.0,发酵时间36 h进行二次发酵。此时发酵效果最好,SOD酶活可达324 U/mL,总酸含量为0.219 g/mL.该圣女果酵素对DPPH自由基的清除能力超过了维C,对羟自由基的清除能力接近维C,但对超氧阴离子的清除能力低于维C。     

Characterization of a New Isolate of Chilli ringspot virus in Yunnan,China

Journal of Crop Science and Biotechnology - Virus diseases are the major limiting factors of chilli production in China. A virus isolate of chilli ringspot virus-Yunnan (ChiRSV-YN) from chilli...     

天津地区番茄褪绿病毒的分子检测和鉴定

为明确2014年夏天在天津番茄种植区采集到疑似感染番茄褪绿病毒的番茄样品的侵染病原,利用To CV外壳蛋白和热激蛋白的特异引物进行反转录PCR(RT-PCR)检测,分别扩增得到特异核苷酸片段。序列分析表明,HSP70核苷酸序列与已登录的番茄褪绿病毒日本分离物Tochigi(AB513442)相似性为99.5%,CP氨基酸和核苷酸序列与已登录的番茄褪绿病毒日本分离物Tochigi(AB513443)相似性分别为99.2%和99.3%,表明天津地区的番茄受到To CV侵染,且天津与日本To CV分离物序列相似性最高。由于调查地区番茄黄化曲叶病毒发生普遍,针对To CV阳性样品利用TYLCV特异性引物进行了分子检测,扩增产物序列与TYLCV以色列株系分离物的相似性均在99.0%以上,表明2种病毒在田间发生复合侵染,该研究首次明确天津地区的番茄受到To CV侵染和To CV与TYLCV的复合侵染。     

番茄斑萎病毒在烟草植株上症状学特征

通过2006年间在美国北卡罗来那州烟草主要生产区的田间调查,系统的描述了番茄斑萎病毒侵染烤烟后,在烤烟叶片上产生的叶片畸形、镶脉、叶脉坏死、坏死斑、同心环纹、叶面皱缩,且整株出现矮化异常生长;将番茄斑萎病毒采用人工摩擦接种到本生烟上,植株被系统侵染,并产生褪绿斑坏死斑、 同心环纹、皱缩等症状,植株一般4～5周内死亡。通过比较两种寄主,发现番茄斑萎病毒侵染两种寄主均出现系统症状,症状相似,但也有不同。     

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F₁ hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F₁ hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.     

A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV)

The reaction to Tomato spotted wilt virus (TSWV) was evaluated in 94 accessions of Nicotiana, originating from the Institute of Soil Science and Plant Cultivation tobacco germplasm collection in Pu?awy, Poland. Tests for resistance were conducted under greenhouse conditions using single TSWV isolate collected from tobacco plantation in Lublin district, Poland. The presence of the virus was verified using DAS-ELISA. SCAR markers associated with TSWV resistance gene were applied. The members of the section Alatae, the genus Nicotiana: N. alata, N. forgetiana, and Nicotiana x sanderae as well as N. tabacum cultivars: ‘Polalta’ and ‘Wiktoria’ with the TSWV resistance gene introduced from N. alata, displayed the hypersensitive reaction (HR) against TSWV (grade 0 on symptom intensity scale). In some of those accessions, the virus spread from the initially infected areas eliciting systemic hypersensitive reaction (SHR). Five accessions of N. alata and three of Nicotiana x sanderae were composed of 6.3–50.0 % of plants in which SHR symptoms appeared. In all of N. forgetiana plants HR reaction was followed by systemic infection (SHR). In N. tabacum ‘Wiktoria’ 21.1 % of plants showed HR reaction, while the remaining were susceptible (S). All of the genotypes which responded with HR or SHR reaction to TSWV infection demonstrated the presence of SCAR markers linked to the resistance gene. The remaining eighty tested accessions were identified as being susceptible upon exposure to TSWV.     

Evaluation of whitefly-mediated inoculation techniques to screen Lycopersicon esculentum and wild relatives for resistance to Tomato yellow leaf curl virus

For two consecutive years nine hybrids and three varieties of tomato, four Lycopersicon peruvianum and four Lycopersicon chilense accessions were screened for Tomato yellow leaf curl virus (TYLCV) resistance. Three inoculation techniques using Bemisia tabaci, the vector of TYLCV, were compared: (1) artificial mass inoculation-simultaneous infection of cultivated and wild material in greenhouses; (2) artificial cage inoculation-individual infection in insect-proof cages; (3) natural field infection. Artificial inoculations led to higher levels of infection, but different patterns of response to each inoculation technique were found depending on the resistance level. Tomato varieties showed an important fruit set reduction after both artificial and natural inoculations. In contrast, field infection was milder in tomato hybrids, in which yield was barely affected. These hybrids showed a wide range of reactions with the two artificial inoculation techniques, but infection was always more severe after mass inoculation. Extreme severity of mass infection made it difficult to differentiate among variable degrees of resistance that were more reliably detected with cage inoculation. The hybrids F3524, F3522, Fiona, and Tyking showed the highest level of resistance. F3524 and F3522 had an acceptable yield in field and cage assays, but their resistance collapsed under massive conditions of infection. Tyking and Fiona exhibited the best response in all conditions, although their yield was moderately reduced in mass assays. Mass inoculation was not adequate for the screening of wild Lycopersicon. Some susceptible plants escaped infection, probably as a consequence of non-preference mechanisms and loss of vector infectivity. Individual inoculation in cages prevented the risk of non-infection, ensuring 100% disease incidence. This technique allowed the selection of highly resistant wild sources. L. chilense LA 1969 and LA 1963 had the highest level of resistance with the three inoculation techniques. L. peruvianum PI-126944 and L. chilense LA 1932, which were only tested in mass and field conditions, also exhibited a promising response. The results proved that the inoculation technique influences the response of tomato and wild Lycopersicon spp to TYLCV. It is concluded that artificial cage inoculation, although more time-consuming, is the most efficient, adequate, and reliable technique to screen both cultivated and wild Lycopersicon species for resistance to TYLCV. This revised version was published online in July 2006 with corrections to the Cover Date.