植物病原菌诱导表达载体构建及在烟草中的瞬时表达研究

根据GenBank中植物病原物诱导型启动子碱基序列设计引物,以烟草基因组DNA为模板,扩增PPP3启动子。用PPP3替换pCAMBIA1301中与gus基因相连的35S启动子,构建重组质粒pCOMBIA ＋PPP3＋gus后导入农杆菌GV3101。通过农杆菌介导的瞬时表达技术将受PPPs控制的gus基因导入烟草。分别接种青枯菌和水杨酸24 h后,烟草叶片中检测到gus基因转录效率分别提高了27．94,17．69倍。表明PPP3启动子具有本底表达水平低、诱导表达活性强的特点。     

衡水湖野生大豆与栽培大豆的RAPD分析

通过对衡水湖野生大豆与栽培大豆分别提取基因组DNA后,采用20个RAPD随机引物对二者进行PCR扩增,经琼脂糖凝胶电泳分析,共扩增出187条带,其中多态条带43条,多态性程度达到23％,说明衡水湖野生大豆与栽培大豆之间存在遗传上的多态性,为下一步进行野生大豆种质资源的保护及利用奠定基础.     

不同O/L值花生品种油酸脱氢酶基因的时空表达特征

本研究以不同油酸/亚油酸比值（O/L比）花生品种为材料,利用实时荧光定量PCR技术,对四个不同O/L花生品种不同组织中油酸脱氢酶基因（FAD2）的表达进行相对定量分析。结果表明：FAD2基因在不同O/L基因型中组成型表达,其在根、茎、叶中表达量低,在花中表达量最高,显著高于荚果等其它组织中的表达量,且品种间差异达显著水平;花U606和花U12在花、幼荚、成熟荚果中的表达量呈依次下降趋势;花育17和狮油红4号在花、幼荚、成熟荚果中的表达量则呈现高-低-次高的表达趋势,差异达显著水平。       

甘蓝型油菜木质素合成关键基因F5H、4CL和COMT的定量表达

以甘蓝型油菜(Brassica napus L )抗倒伏品种浙平1号和易倒伏品种高芥1号为材料,采用荧光定量PCR方法对木质素合成关键基因F5H（阿魏酸-5-羟基化酶）、4CL（4-香豆酸CoA连接酶）和COMT（咖啡酸/5-羟基阿魏酸-O-甲基转移酶）在薹期和角果期根颈部、茎部的相对表达量进行了分析。结果显示,F5H在薹期和角果期根颈部的相对表达量,易倒伏材料高于抗倒伏材料;茎部的相对表达量,抗倒伏材料高于易倒伏材料。4CL和COMT基因在薹期根颈部、茎部和角果期根颈部的相对表达量,易倒伏材料高于抗倒伏材料;角果期茎部的相对表达量,抗倒伏材料显著（P＜0.05）高于易倒伏材料。结果表明,油菜角果期茎部木质素合成关键基因F5H、4CL和COMT的相对高量表达可能有助于增强植株的抗倒伏能力。     

茶尺蠖核型多角体病毒(EoNPV)实时荧光定量PCR检测方法的优化及应用

选用茶尺蠖核型多角体病毒(EoNPV)基因组中序列特异性很高的p16基因为目标基因,通过设计引物、PCR扩增、目的片段与载体连接转化获得含p16基因的重组质粒进行测序,进而以经测序鉴定的p16基因的PCR纯化产物作为实时荧光定量PCR标准品模板,稀释后建立SYBR Green I荧光定量标准曲线,统计分析显示标准品浓度的对数值与Ct值之间存在良好的线性关系(R2=0.9981)。该方法的检测灵敏度为102,扩增产物形成单一的特异性熔解峰,组内组间变异系数均小于3%。同时分别对含有其他茶树害虫病毒和不同浓度的茶尺蠖病毒产品的样品进行检测,能明确样品中是否含有EoNPV及含量。根据已建立的方法对感染病毒后不同时间段的茶尺蠖幼虫进行检测,每克幼虫样本内p16基因拷贝数的增殖倍数对数值与感染时间呈正相关(R2=0.9935),可以获得茶尺蠖幼虫感染病毒后不同时间的增殖动态。上述结果表明,该方法能定性定量鉴定茶尺蠖核型多角体病毒,可用于茶尺蠖核型多角体病毒类生物农药的检测和感染过程检测。     

Developmental Responses of Wheat cv. Norin 61 to Fluence Rate of Green Light

Summary

Wheat (Triticum aestivum L. cv. Norin 61) plants were grown under five different photon flux densities obtained by modulating the number of green light-emitting diodes (LEDs) under white fluorescent lamps. The experiment was conducted under continuous irradiation at a constant temperature of 20°C to clarify the developmental responses of wheat to the fluence rate of green light. The higher the photon flux density of green light, the shorter the number of days from emergence to heading. The earliest heading was observed in the plants grown under 496 green LEDs, 32.0 days after emergence. A significant logarithmic function could fit the relationship between the fluence rate of green light and developmental rate. In this report, principal component analysis (PCA) was adopted to analyze the confounding of green light versus photosynthetic photon flux density (PPFD) with 17 developmental and morphological traits. The eigenvalues explained were 5.64, 3.20, and 2.61, respectively, for the first, second and third principal components (PCs). The first PC was assumed as the factor related to the isometric growth, and the third PC was assumed as the factor related to the developmental rate and culm elongation. Therefore, it was supposed that the first and third PCs were affected by the PPFD and the photon flux density of green light, respectively. The results suggested that the fluence rate of green light affects the development of wheat as a signal source. Furthermore, the development of wheat was promoted by the green light independent of PPFD.     

针对fimW基因沙门氏菌的特异性PCR检测

为建立沙门氏菌快速检测方法,本研究根据沙门氏菌Ⅰ型菌毛蛋白调控基因fimW设计一对引物,建立PCR检测方法,并对收集的A群～F群14个血清型40株沙门氏菌和5种12株非沙门氏菌菌株进行PCR扩增.结果显示所有沙门氏菌均扩增出与预期(477 bp)相符的特异性片段,而非沙门氏菌扩增结果均为阴性.另外,以肠炎沙门氏菌50336株DNA为模板,敏感性试验结果显示该方法可以检测出扩增体系中1pg DNA染色体和102cfu的沙门氏菌.表明本研究建立了检测沙门氏菌简洁、敏感、特异的PCR方法.     

鸭源鸡杆菌双重PCR检测方法的初步建立

为建立鸭源鸡杆菌(G.anatis)双重PCR检测方法,并对鸡杆菌分离株进行种类鉴定,本研究根据GenBank中G.anatis12656-12株的gtxA基因序列设计一对PCR引物,合成扩增rpoB基因的引物作为内参基因,建立了G.anatis双重PCR检测方法.检测结果显示:以G.anatis Yu-PDS-RZ-1-SLG株DNA为模板进行的双重PCR检测能够扩增出两条目的片段;其它16株细菌包括副鸡禽杆菌、多杀性巴氏杆菌、大肠杆菌、沙门氏菌、福氏志贺菌和奇异变形杆菌均只扩增出了一条目的片段;该方法可以检测到浓度为6.5×102 cfu/mL的G.anatis;34株鸡杆菌分离株均扩增出了两条预期大小的目的片段.此外,序列分析结果表明,10株鸡杆菌河南分离株的rpoB基因序列与鸭源鸡杆菌种参考株(CCUG15563)的同源性最高(97.5％～99.0％),属于鸭源鸡杆菌种.本研究建立的G.anatis双重PCR检测方法,可用于含gtxA基因鸡杆菌菌株的鉴定及临床病原学诊断.     

山羊和绵羊嗜血支原体PCR检测方法的建立与应用

为建立羊嗜血支原体(M.ovis)病快速检测方法,本实验根据GenBank中登录的羊M.ovis 16S rRNA基因序列设计一对引物,以山羊和绵羊M.ovis基因组DNA为模板,建立M.ovis PCR检测方法,并进行特异性、敏感性及临床应用试验。结果显示,建立的M.ovis PCR检测方法扩增片段大小为508 bp,与GenBank中M.ovis参考株同源性达99%以上,该方法与猪嗜血支原体、牛嗜血支原体等病原体无交叉反应,最低检测40个拷贝的DNA;通过对延边地区60份山羊和绵羊血液样本的检测结果表明,建立的PCR检测方法具有特异、敏感、准确等优点,完全适用于M.ovis的检测。     

Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes

The hippocampus is affected by various stimuli that include hyperglycemia, depression, and ischemia. Calcium-binding proteins (CaBPs) have protective roles in the response to such stimuli. However, little is known about the expression of CaBPs under diabetic conditions. This study was conducted to examine alterations in the physiological parameters with type 1 diabetes induced with streptozotocin (STZ) as well as time-dependent changes in the expression of two CaBPs changes of were being evaluated. Rats treated with STZ (70 mg/kg) had high blood glucose levels (>21.4 mmol/L) along with increased food intake and water consumption volumes compared to the sham controls. In contrast, body weight of the animals treated with STZ was significantly reduced compared to the sham group. CB-specific immunoreactivity was generally increased in the hippocampal CA1 region and granule cell layer of the dentate gyrus (DG) 2 weeks after STZ treatment, but decreased thereafter in these regions. In contrast, the number of PV-immunoreactive neurons and fibers was unchanged in the hippocampus and DG 2 weeks after STZ treatment. However, this number subsequently decreased over time. These results suggest that CB and PV expression is lowest 3 weeks after STZ administration, and these deficits lead to disturbances in calcium homeostasis.