用焦磷酸测序技术检测凡纳滨对虾组织蛋白酶基因单核苷酸多态性

焦磷酸测序是一种新型的DNA测序技术,近年来开始应用于单核苷酸多态性(SNPs)检测.组织蛋白酶基因(Cathepsin-L,CTSL)在对虾的蜕皮周期中起重要的作用.本研究利用焦磷酸测序技术,对96个凡纳滨对虾的CTSL基因序列(GnBank登录号:AY366355)的C681G SNP位点进行检测分型.结果发现C/C、C/G和G/G基因型,频率分别是0.81、0.16和0.03,频率分布符合Hardy-Weinberg平衡.统计分析结果表明在该群体中C681G SNP和体重关系不显著(P>0.05).研究结果显示焦磷酸测序技术是一种高效的SNP检测技术,可以在对虾的遗传研究中发挥重要作用.     

中国大麦黄矮病毒及 BYDV-GAV 株系研究进展

大麦黄矮病毒（Barley yellow dwarf virus, BYDVs）隶属于黄症病毒科（Luteovirdae）黄症病毒属（Luteovirus），由该病毒引起的小麦黄矮病最早在美国发生并报道，此后在国外其他地域也曾有过发生报道。在我国，小麦黄矮病最早于 1960 年在陕西、甘肃发生并报道，近年来，在我国其他地区也有发生，该病害被称为麦类作物上的“癌症”，对麦类作物生产影响较大，为害严重时，造成麦类作物产量大幅降低。BYDV 株系的划分依据为其传毒介体蚜虫的传播专化性，即一个病毒株系只能由一种或两种蚜虫进行传播，根据 ICTV 国际病毒委员会分类系统报道，目前国际上确定的该病毒株系有 BYDV-MAV、BYDV-PAV、BYDV-RMV、BYDV-SGV 等，我国鉴定有 BYDV-PAV、BYDV-GPV、BYDV-GAV、BYDV-RMV 4 个株系，GAV 为我国流行株系。主要从 BYDV 的为害症状、株系分化、传播介体、基因组结构和功能、病害防治方面进行相关综述，着重介绍 BYDV-GAV 株系的抗性鉴定、抗性基因等方面的相关研究进展，以期为该病害的抗病育种和防治提供一定的理论基础。     

OsI2基因的克隆及其植物表达载体的构建

在旱稻IRAT109干旱胁迫下cDNA芯片分析结果的基础上,通过RT-PCR,5'-RACE及3'-RACE方法,从IRAT109总RNA中扩增得到了干旱胁迫下芯片表达谱中上升表达强度第二的基因全长序列,命名为OsI2,全长有523 bp,并对基因序列结构进行了分析.该基因与全长cDNA文库中的CT836140.1有99%同源.OsI2基因编码产物对应1个包含57个氨基酸的开放阅读框(ORF),为一功能未知蛋白.其编码产物也可能对应两个中间相隔19 bp的较小ORFs(43个氨基酸和42个氨基酸),都是功能未知的蛋白.在pBI121载体的基础上,将OsI2基因与CaMV35S连接成功构建了pBl121-OsI2植物表达载体,为进一步研究其功能创造了条件.     

牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。     

莱航鸡rDNA重复基因家族的克隆

动物rDNA基因是一种GC含量较高、结构复杂的重复序列。通过结合生物信息学技术,经反复摸索后选用LAPCR法扩增莱航鸡rDNA基因重复序列,经测序鉴定最终克隆了莱航鸡的3个rDNA基因及其2个间隔序列。研究对克隆复杂DNA序列时引物设计的特别规则、反应体系的改进、DNA聚合酶的选用、循环参数的调整等进行了探索。     

鸡传染性喉气管炎病毒姻台株gB基因的序列测定及其结构功能分析

研究以鸡传染性喉气管炎病毒（ILTV）烟台株为模板，PCIK扩增出1条约2．7的gB基因片段，并将其克隆到pMD18-T载体中。序列测定显示，gB基因长2622bp，包含完的阅读框架。序列比较分析发现，烟台株和王岗株、河北株的班基因核苷酸序列与SA2株的比均在第89位上缺失1个碱基G，而在第102位核苷酸处插入1个碱基A，从而引起氨基酸序中第29-32位5个氨基酸的移码突变。烟台株还有另外7个碱基发生突变，使得其班基因与北株、王岗株、SA2疫苗株的gB基因核苷酸同源性分别为99．8％、99．7％和99．6％，氨基酸的同性分别为99．4％、99．4％、98．9％，表明不同ILTV毒株之间gB基因是非常保守的。划型相列河源     

The potential role of PR-8 gene of apple fruit in the mode of action of the yeast antagonist,Candida oleophila,in postharvest biocontrol of Botrytis cinerea

Several pathogenesis-related (PR) genes of apple have been cloned and their response to different pathogens has been studied. Different PR genes, however, may have a variable response depending on the specific organ or tissue as well as microbe. The objective of the current study was to characterize the expression of specific apple PR genes in fruit tissues in response to the antagonistic yeast, Candida oleophila (a common postharvest biocontrol agent), and the fungal pathogen, Botrytis cinerea (a major postharvest pathogen). Apple PR-5 and PR-8 gene expression was characterized in fruit in response to C. oleophila and B. cinerea. Results indicated that PR-8 expression was significantly elevated in response to both fungi. In contrast, neither C. oleophila nor B. cinerea treatment markedly affected PR-5 expression. The PR-8 gene was then synthesized and cloned into a Pichia pastoris expression system to study the antifungal activity of the PR-8 protein against B. cinerea both in vitro and in vivo. Collectively, the PR-8 gene of apple is associated with the response to B. cinerea infection, and may play a role in the mechanism by which C. oleophila effectively inhibits B. cinerea disease in apple fruit, namely by the induction of this specific host PR gene.     

Proteolytic activity in soil: A review

The aim of this work is to review current knowledge on inputs, sources and regulation of protease activities in soils from different ecosystems, while exploring limitations to proteolysis and N mineralisation. Extracellular proteases enter the soil via microbial production and other sources, including plant root exudates, animal excrements, decomposition processes and leaching from agro-industrial fertilisers. The synthesis and activities of proteases in soil are regulated by many factors, including climate, soil properties and the presence of organic compounds of plant and microbial origin. Two particularly important areas for future research are the regulation of proteolysis by low-molecular-weight organic compounds, including amino acids, sugars, flavonoids, plant hormones and siderophores, as well as the identification and characterisation of proteinaceous protease inhibitors of plant and microbial origin in the soil. Despite all the work that has been performed on soil proteases, our understanding of the roles of extracellular plant root proteases in N nutrition is weak. Furthermore, the regulation of soil proteolytic activities of different ecosystems, especially in terms of pollutant inputs and the impact of climate change, requires investigation. Other areas that pose important questions for the future include assessments of protease inhibitor inputs to the soil, regulation of these inhibitors via naturally occurring soil organic compounds and the interactions between soil organisms.     

韭菜迟眼蕈蚊紫外敏感视蛋白基因的克隆及光强度对其表达量影响

为明确韭菜迟眼蕈蚊Bradysia odoriphaga紫外敏感视蛋白基因Bo-uv的作用及其与趋光性的关系,利用常规PCR方法克隆获得Bo-uv基因的全长cDNA序列,分析了其敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白氨基酸序列之间的系统进化关系,运用qPCR技术检测了不同发育阶段、不同组织及不同光强度下Bo-uv基因的相对表达量。结果表明,Bo-uv基因cDNA全长2 757 bp,开放阅读框1 542 bp,编码514个氨基酸。韭菜迟眼蕈蚊紫外敏感视蛋白的氨基酸序列与其它12种昆虫同源蛋白的氨基酸序列一致性为21.93%～43.00%,与橘小实蝇Bactrocera dorsalis的氨基酸序列同源性最高。Bo-uv基因在韭菜迟眼蕈蚊蛹末期、成虫期表达,在成虫头部的相对表达量较高。在0～10 000 lx光强范围内雌、雄成虫体内该基因的相对表达量均呈先增高后降低趋势。与对照相比,1 000 lx光强度下其相对表达量显著升高,10 000 lx时相对表达量显著降低。表明光强度能够有效地调控Bo-uv基因的表达,该基因在韭菜迟眼蕈蚊感知外界光刺激过程中具有重要作用。     

云南省元阳县籼/粳型稻瘟病菌无毒基因Avr-Pia、Avr-Pita1和Avr-Pii多样性分析

为明确云南省元阳县籼/粳型稻瘟病菌中无毒基因的多样性及差异,采用接种鉴别品种方法和PCR技术对9株粳型菌株和21株籼型菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的多样性进行分析。结果显示,30株稻瘟病菌菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的平均出现频率分别为33.3%、76.7%和0,Avr-Pia、Avr-Pita1在籼型菌株和粳型菌株中的出现频率分别为4.8%和100.0%、66.7%和100.0%。Avr-Pia主要为存在/缺失的多样性,籼/粳型菌株对水稻品种Achiasahi(Pia)的致病率分别为100.0%和11.1%;Avr-Pita1可划分为3个类群,除菌株CH1195属于PO类群外,籼型菌株均属于J1类群,粳型菌株则属于J2/J3类群;籼/粳型菌株对水稻品种K1(Pita)的致病率分别为100.0%和0,且籼/粳型菌株Avr-Pita1蛋白序列在第83位和第192位氨基酸差异明显。Avr-Pii在30株稻瘟病菌菌株中均未检出,籼/粳型菌株对水稻品种Fujisaka No. 5(Pii)的致病率分别为100.0%和0。表明Avr-Pia和Avr-Pita1在粳型菌株中的出现频率高于籼型菌株且变异程度低,粳型菌株中可能存在其它无毒基因使其不能侵染Fujisaka No. 5。