甘蓝型油菜花序长发夹ｌｈＲＮＡｉ文库的构建

异源四倍体油菜含有大量多拷贝基因及冗余基因,难以通过T-DNA插入、物理及化学诱变等常规突变体库创制方法发掘功能基因。为开发适应于油菜的简单高效的突变体库创制方法,本研究通过滚环复制方法构建了甘蓝型油菜的花序lhRNAi文库,并对这种新型的干扰文库进行质量评估;然后将该花序lhRNAi文库对甘蓝型油菜进行遗传转化,获得763株T0植株,从中鉴定分离出74株具有可见表型变异的花发育相关突变体,包括花瓣减少、形状异常,柱头卷曲,雄蕊退化萎缩,雄性不育（不能产生花粉）、死蕾或花蕾闭合等。说明通过滚环复制介导的lihRNAi文库的构建方法可以成功应用到油菜中,这将为油菜花序发育相关基因功能的研究提供重要的研究平台。     

生鲜牛乳中产肠毒素大肠埃希菌环介导等温扩增技术的建立与应用

为了快速检测生鲜牛乳中的产肠毒素大肠埃希菌(enterotoxigenic E.coli,ETEC),防止食源性产肠毒素大肠埃希菌给人类造成的危害,建立快速检测不耐热型产肠毒素大肠埃希菌的环介导等温扩增方法(LAMP)。敏感性和特异性试验结果表明,该方法敏感性高,特异性强,能够从生鲜牛乳中检测到1pg产肠毒素大肠埃希菌DNA,与其他型大肠埃希菌及细菌不发生交叉反应。利用建立的LAMP技术对来自西宁市奶牛场及自由市场的21份生鲜牛乳样品进行检测,6份样品呈阳性,阳性检出率为28.6%,从分子水平为生鲜牛乳中产肠毒素大肠埃希菌的检测提供了一种快速、准确的手段,对保障生鲜牛乳的食品安全有重要意义。     

肝螺杆菌LAMP快速检测方法的建立及初步应用

根据肝螺杆菌fla B基因保守区域设计一组特异性引物,经过条件优化、特异性和敏感性分析,成功建立了肝螺杆菌LAMP快速检测方法。结果显示,建立的LAMP扩增方法仅能检测出肝螺杆菌,其他常见细菌未见特异性扩增。检出肝螺杆菌最低模板量为86.9fg/L,是普通PCR所需模板量的1/10。本研究建立的LAMP快速检测方法具备操作简单、特异性好、灵敏度高的优点,结果易于观察,对仪器设备要求低,适宜推广应用。     

中国樱桃S-RNase基因PCR扩增技术体系的建立

通过对TaqDNA聚合酶、Mg2+、dNTP、通用引物、模板DNA浓度等反应参数的系统研究,建立了中国樱桃S-RNase基因特异PCR扩增体系。该体系反应的总体积25μL,其中Taq酶1.25U,MgCl22.5mmol/L,dNTP0.15mmol/L,通用引物0.25μmol/L,模板DNA 50 ng。反应程序为:94℃预变性3 min;33个循环的94℃变性1 min,56℃退火45 s,72℃延伸1.5 min;最后72℃延伸10 min。利用优化的扩增体系在中国樱桃的不同品种中获得有效扩增,进一步证明了该体系具有良好的稳定性和重复性。     

DNA amplification fingerprinting and marker screening for pseudo-testcross mapping of flowering dogwood ( Cornus florida L.)

DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers was used to study genetic relationships between cultivars of flowering dogwood (Cornus florida L.), evaluate extent of plant hybridization, and generate markers in pseudo-testcross mapping at the intraspecific level. Modified Taguchi optimization methods defined a robust DAF system based on high annealing temperature (48–52 °C) and primer concentration (typically 8 μM) that was used to study genetic diversity of representative dogwood cultivars and hybrids. Phenetic analysis using cluster and numerical methods showed that: (1) cultivars were relatively conserved at the genetic level; (2) their hybridization could be identified in the F1 progeny in the absence of phenotypic or physiological markers; (3) several cultivars grouped according to their recorded ancestry; and (4) dogwood anthracnose-resistant lines originally selected in Catoctin Mountain Park (Maryland) grouped separately from those of southern origin. The DAF protocol was also tested in pseudo-testcross mapping of dogwood at the intraspecific level. A preliminary screening of parents ‘Pink Sachet’ and ‘Fragrant Cloud’ and 7 F1 segregants with 22 octamer primers produced 703 amplified loci, 30 and 39 of which were male and female markers segregating at 1:1 ratios with 98.6% confidence levels in pseudo-testcross configuration. Overall results show that DAF generated markers very efficiently (3 per primer) despite the close relatedness of parental dogwood cultivars. This study constitutes the basis for a future genetic linkage mapping and marker-assisted selection (MAS) effort initially targeted to control important fungal diseases in dogwood. This revised version was published online in July 2006 with corrections to the Cover Date.     

长发带芒草y型高分子量谷蛋白亚基的鉴定与分析

为了证实长发带芒草中的y型高分子量谷蛋白亚基的存在,利用SDS-PAGE分析了3份长发带芒草的高分子量谷蛋白亚基组成,发现其y亚基的迁移率均较普通小麦中迁移率最快的D y12亚基迁移率更快,应用PCR扩增、序列测定及基因编码区体外表达等方法研究了1份材料中的y亚基,确认了长发带芒草比普通小麦中迁移率最快的D y12亚基迁移率更快的T ay亚基的真实存在及表达。研究结果证实带芒草属具有与普通小麦中相类似的y型高分子量谷蛋白亚基。     

Molecular detection of insecticide resistant alleles

The summary discusses techniques used to investigate mechanisms of resistance in the Colorado potato beetle (Leptinotarsa decemlineata) to several classes of pesticides.     

十足目虹彩病毒(DIV1)环介导等温扩增(LAMP)检测方法的建立及应用

本研究以十足目虹彩病毒(Decapod iridescent virus 1, DIV1)主要衣壳蛋白基因为靶序列设计引物,建立了DIV1的环介导等温扩增(Loop-mediated isothermal amplification, LAMP)检测方法,并以pMD18-DIV1质粒标准品为模板对该方法的检测灵敏度、检测特异性等进行了评估。结果显示,此方法最适反应温度为64.4℃,优化后的25 μl反应体系中包含2.5 μl 10×Isothermal amplification buffer、4.0 mmol/L Mg2+、1.2 mmol/L dNTPs、6.4 U Bst 2.0 WarmStart® DNA聚合酶、0.8 μmol/L EvaGreen®和4.4 μl ddH2O。该方法检测灵敏度下限为3.54×102拷贝/反应;与虾肝肠胞虫(EHP)、致急性肝胰腺坏死病副溶血弧菌(VpAHPND)、对虾偷死野田村病毒(CMNV)、传染性皮下及造血组织坏死病病毒(IHHNV)、白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)和黄头病毒(YHV)等主要虾类病原没有交叉反应;具有较好的重复性和稳定性。以GeneFinder®替换EvaGreen®并将其预置于反应管内,结合上述扩增方法可实现对DIV1的现场快速高灵敏检测。本研究建立的DIV1-LAMP实时荧光定量和现场检测方法具有灵敏、特异和快速等特点,为近几年新发虾类病原DIV1的定性、定量以及现场快速检测提供了新的技术选择,有利于对虾养殖业中开展DIV1的监测、预警和防控。     

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum . LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE , of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 × 10¹ to 2.0 × 10⁹ copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.     

PCR法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA

4 香豆酸 :CoA连接酶 (4CL)是木质素生物合成过程中重要的酶 .该文利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法直接获得了紫穗槐 4CLAcDNA全长序列 ,避免了复杂的构建、筛选cDNA文库的过程 .先用简并PCR得到了 4CLA1片段 ,又据此片段设计反向嵌套引物 ,用RACE方法获得未知的 5′和 3′端序列 .所获得的全长 4CLAcDNA ,编码 5 40个氨基酸 .氨基酸序列同源性分析表明4CLA是典型的 4CL蛋白 ,含有预计的AMP binding位点、催化反应区和保守的Cys .