水稻条叶枯病毒基因产物在水稻和昆虫体内的Western印迹分析

将AStV基因组组分3编码的NS3和NC蛋白基因以及组分4编码的NCP和NSvc4蛋白基因分别克隆于大肠杆菌表达载体pGEX3X在IPTG诱导下,表达出4种融合蛋白。这些表达产物用于免疫动物产生多克隆抗血清。以制备的多克隆抗血清为探针,Western印迹分析了宿主植物和介体昆虫体内NS3、NC、NCP和NSvc4种基因表达的产物。NC蛋白的抗体可在病株和病毒粒子制备物中以及NCP抗血清仅在病株中检测到相应大小的产物,其余的均与预期值相停。     

单头灰飞虱体内水稻条纹叶枯病毒的快速检测

利用RT-PCR检测感病植株和单头带毒灰飞虱, 均扩增出水稻条纹叶枯病毒特有的一个长540 bp的片段, 且人工饲毒虫的病毒含量高于自然感病稻田虫。以病毒S蛋白的多克隆抗血清, 采用Western印迹技术从单虫体内检测到病毒抗原的2个专一性条带, 大小分别为20.7和19.7 kDa。DIBA (dot immunobinding ass ay)检测法快速、简便, 但专一性和灵敏度不及RT-PCR与Western印迹检测。对不同检测方法所得昆虫带毒率差异的原因进行了探讨。     

受NaCl诱导的盐藻果糖-1,6-二磷酸醛缩酶cDNA的克隆及其在烟草中的表达

采用快速扩增cDNA末端(RACE)技术, 获得了盐藻(Dunaliella salina)受诱导表达的果糖-1,6-二磷酸醛缩酶全长cDNA (DsALDP). 蛋白质序列分析发现, DsALDP与许多植物叶绿体的果糖-1,6-二磷酸醛缩酶(AldP)具有较高的同源性(66%~73%). 系统进化分析进一步证明DsALDP与藻类的AldP亲缘关系最近. 就表达谱而言, DsALDP是NaCl诱导下新表达的转录本, 其表达水平随诱导时间的不同而呈显著变化. 筛选的DsALDP cDNA构建到双元载体pBI121并导入农杆菌用以转化烟草, 对4个T₁代转基因植株进行Southern检测, 证明DsALDP已整合入转基因植株的基因组. RT-PCR分析显示DsALDP在这些植株中均得到有效表达. 生物测试表明, T₁-1, T₁-2和T₁-3植株在100~200 mmol/L NaCl下仍保持醛缩酶活性, 且其脯氨酸含量均有不同程度的升高.     

不同SSR标记检测技术及其在花生栽培种遗传多样性分析中的应用

以85份花生栽培种为材料,分别应用银染法和荧光检测法检测9对SSR引物的扩增产物。比较结果显示,荧光检测法具有灵敏度高、检测结果准确、效率高等优点。聚类分析表明,银染法与荧光检测法分别能够区分74个、82个花生品种,并分别聚成8个、9个类群;荧光检测法的聚类结果虽然反映的品种间遗传多样性较低,但与品种类型、产地及其亲缘关系相关程度更高,表明荧光检测数据更精确、可靠。遗传多样性分析发现,地方品种的遗传多样性指数最高,其次为多粒型育成品种,表明我国地方品种和多粒型育成品种蕴藏了丰富的优异性状,有利于对其挖掘和利用。     

单头灰飞虱体内水稻条纹叶枯病毒的快速检测

利用RT-PCR检测感病植株和单头带毒灰飞虱,均扩增出水稻条纹叶枯病毒特有的一个长540 bp的片段,且人工饲毒虫的病毒含量高于自然感病稻田虫。以病毒S蛋白的多克隆抗血清,采用Western印迹技术从单虫体内检测到病毒抗原的2个专一性条带,大小分别为20.7和19.7 kDa。DIBA（dot immunobinding assay）检测法快速、简便,但专一性和灵敏度不及RT-PCR与Western印迹检测。对不同检测方法所得昆虫带毒率差异的原因进行了探讨。     

Cloning of a NaCl-induced fructose-1, 6-diphosphate aldolase cDNA from<Emphasis Type="Italic">Dunaliella salina</Emphasis> and its expression in tobacco

Using Rapid Amplification of cDNA ends (RACE) technique, the full-length cDNA encoding a NaCl-induced fructose-1, 6-diphosphate aldolase (DsALDP) was obtained. It was shown that the DsALDP had a relatively high homology (66%–73%) to chloroplast fructose-1, 6-diphosphate aldolase (AldP) in many plants according to their amino acid sequences. The phylogenetic analysis further confirmed that AldP in alga is the nearest to DsALDP. As to its expression pattern,DsALDP was denovo synthesized by NaCl induction. Its expression level was significantly changed with inducing time. After the selectedDsALDP cDNA subcloned into a binary vector pBI121, the new construct was introduced into tobacco byAgrobacterium tumefaciens. The results of Southern blot and RT-PCR analysis of four transgenic T1 plants indicated thatDsALDP was integrated into genome of these transgenic plants and effectively expressed. Aldolase activities have been detected in T1-1, T1-2 and T1-3 plants by bioassay under 100–200 mmol/L NaCl. It was also observed that proline contents in them were differentially increased.