瘤胃脂肪分解菌实时荧光定量PCR方法的构建

为定量测定瘤胃脂肪分解菌,试验构建了脂肪分解菌实时荧光定量PCR的标准品及标准曲线。提取瘤胃微生物总DNA,以脂肪分解菌16S r DNA特异性引物进行PCR扩增,回收PCR产物,与PMD19-T Vector连接并转化大肠杆菌。阳性重组质粒经PCR和测序鉴定后,将提取的质粒DNA进行梯度稀释并作为模板,利用荧光定量PCR反应做出标准曲线。结果显示:PCR产物与目的基因的相似性大于99%,以不同稀释度的重组质粒为模板获得的荧光定量PCR扩增曲线差异明显,构建了相关系数接近1的标准曲线,且熔解曲线峰值单一。试验建立的实时荧光定量PCR方法可用于瘤胃脂肪分解菌的定量检测,为进一步研究该菌在反刍动物瘤胃发酵中的分子机理奠定了基础。     

玉米样品中转基因玉米Bt176含量检测

根据其检测特异性,转基因产品检测技术可分为筛选、基因、构建和转化体特异性检测四类.由于转化体特异性检测方法的具有高度特异性,目前,一些发达国家对于转基因产品检测方法正逐步过渡到转化体特异性序列的检测方法.本研究针对转基因玉米Bt176的外源基因3'端与玉米基因组DNA相接区序列设计具有转化体特异性的引物和荧光探针.利用实时荧光定量PCR,以已知Bt176含量样品为标准样品建立内外源基因标准曲线,利用该标准曲线对多个玉米样品Bt176玉米成分含量进行检测,结果显示该方法检测结果准确,检测特异性强,Bt176玉米检测极限浓度达到0.001 ng/μL.同时该方法操作简单,适合转基因样品大批量检测.     

Nitrogen availability decreases prokaryotic diversity in sandy soils

In this study, the interrelation between nitrogen availability and prokaryotic diversity are studied using a well-characterised system from a long-term field experiment on a loamy sandy soil. The prokaryotic potential functional diversity and community composition were assessed using community-level physiological profiling (CLPP), and their phylogenetic diversity was analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing analysis. Highest prokaryotic potential functional diversity was measured in the control soil receiving no N fertilisation, indicating an efficient as well as versatile utilisation of the substrates in this soil. Both substrate utilisation richness and substrate utilisation evenness, the two constituents of the functional diversity, were decreased with increasing N supply. Furthermore, distinct prokaryotic community compositions were generated in N-enriched soils compared to unfertilised control soils. These differences suggest a dominance of populations adapted to utilising readily available substrates. We demonstrated that the shift in prokaryotic functional diversity was connected to a shift in the phylogenetic structure of the bacterial and archaeal communities. Taken together, our data clearly show that, for the sandy soil system, prokaryotic diversity and N availability were interrelated.     

Combined effect of fertilizer and herbicide applications on the abundance, community structure and performance of the soil methanotrophic community

The use of agrochemicals, such as mineral fertilizers and herbicides in agricultural systems, may affect the potential of soils to act as a sink for methane. Typically, the effect of each agrochemical on soil methane oxidation is investigated separately whereas in the field these agrochemicals are used together to form one comprehensive land management system. Here we report the results of field experiments that assessed the combined effect of multiple fertilizer and herbicide (nicosulfuron, dimethenamide and atrazine) applications on the soil methanotrophic community. Soils treated with organic fertilizer had three times higher methane oxidation rates compared to soils receiving mineral fertilizers. These higher oxidation rates were positively reflected in a significantly enhanced abundance of methanotrophs for the organic fertilized soils. In contrast, herbicide application did not alter significantly the soil methane oxidation rate or the methane-oxidizing population abundance. Subsequently, the methanotrophic community structure was analyzed with group-specific DGGE of 16S rRNA genes. Cluster analysis of the methanotrophic patterns clearly separated the mineral from organically fertilized soils. Less pronounced clustering differentiated between chemical and manual weed control. Furthermore, cluster analysis of the methanotrophic community revealed that soil type was the primary determinant of the community structure. Our results indicate that fertilizer type had the greatest influence on methane oxidizer activity and abundance. Soil type had the most pronounced effect on the microbial community structure.     

陆地棉(G. hirsutum L.) N-乙酰氨基葡萄糖转移酶基因(GhGnT)的克隆及耐盐性功能分析

在耐盐相关基因芯片基础上,我们筛选到盐诱导显著上调表达(Ratio>2)的N-乙酰氨基葡糖转移酶基因.选取耐盐材料中9806为材料,根据耐盐性抑制消减文库EST序列设计引物,利用RACE及RT-PCR技术获得该基因cDNA全长1970 by,命名为GhGnT.通过Blast比对,发现该基因与蓖麻乙酞氨基葡萄糖转移酶基因...     

利用共享对象实现基于Web的远程教学实时课件

本文介绍了Macromedia Flash Media Server(FMS)服务器平台中的共享对象的概念,通过实例详细描述了如何利用共享对象制作实时教学课件,并应用在基于Web的远程教学软件之中.     

实时实地氮肥管理对水稻产量和稻米品质的影响

【目的】探讨实时实地氮肥管理对水稻产量和品质的影响。【方法】试验于2004和2005年在大田条件下，以两优培九、汕优63为材料研究了不同叶绿素仪（SPAD）预设阈值指导下的实时实地氮肥管理方式的产量与产量形成以及相应的稻米品质特性。【结果】在实时氮肥管理（RTNM）模式下，两优培九和汕优63各施氮处理比不施氮处理增产幅度分别达21.12%～57.65%和15.00%～31.18%。在实地氮肥管理（SSNM）模式下，两优培九和汕优63 SSNM处理比不施氮小区增产幅度分别达45.44%～50.71%和28.53%～32.40%。两优培九SPAD阈值分别由34～45的RTNM模式下，当SPAD阈值介于38～41之间时（氮肥用量120～165 kgN•ha-1）可以改善稻米的外观品质和加工品质；汕优63则以SPAD阈值36～39（氮肥用量：120～165 kgN•ha-1）范围内有利于改善米质。SSNM模式下以SPAD施肥阈值为37-39（氮肥用量130 kgN•ha-1）进行氮肥运筹能显著改善两优培九的加工品质、外观品质和营养品质；SSNM模式下汕优63以SPAD阈值为35～37时稻米品质相对较好，同时产量也比较高。【结论】实时实地氮肥管理能较好地协调水稻产量和品质的关系，关键措施是依据品种特性确定适宜的预设SPAD阈值。在本试验条件下，实时实地氮肥管理模式两优培九以SPAD 38～39、汕优63以SPAD 35～37左右时能获得较高的产量和部分地改善米质，可以作为生产上应用实时实地氮肥管理时的推荐阈值。     

用实时荧光PCR方法定量检测Bt176转基因玉米

以实时荧光PCR技术鉴定商业化种植的转基因玉米Bt176品系。根据转基因玉米Bt176中内源基因Zein和外源基因cry1Ab设计引物及TaqMan荧光探针,成功建立了检测转基因玉米Bt176品系的实时荧光PCR方法。结果表明,应用实时荧光PCR方法检测转基因作物,不仅可以达到品系鉴定的作用,而且可以用于转基因成分的检测。该方法简便迅速,降低了污染机会,为转基因作物及产品的品系检测提供了新方法。     

板栗赤霉素缺陷型短雄花序芽变的初步鉴定及CmGID1基因的表达分析

 利用赤霉素、细胞分裂素、脱落酸、乙烯利和吲哚乙酸分别涂抹板栗短雄花序,结果显示仅赤霉素能够抑制短雄花序顶端的细胞程序性死亡,并在一定程度上恢复短雄花序的伸长生长。通过RT-PCR扩增方法,从板栗短雄花序芽变和正常雄花序cDNA中克隆出赤霉素受体基因GID1部分序列。测序及序列分析结果表明：克隆的cDNA片段为786 bp,所推导的氨基酸序列与杨毛果GID1氨基酸序列有91%的同源性,与陆地棉、蓖麻GID1均有85%的氨基酸序列同源性。经氨基酸序列功能分析,推测该GID1序列是板栗有编码功能的赤霉素受体基因,命名为CmGID1（GenBank登录号为HQ651231）。实时荧光定量PCR结果显示,从花序萌发至花药萌发前期,除其中一个时期（5月23日）外,芽变短雄花序CmGID1的表达量均高于正常雄花序;在花序的快速生长期,芽变短雄花序CmGID1表达量显著高于正常雄花序（P < 0.01）;且赤霉素处理后恢复生长的短雄花序CmGID1表达量显著降低（P < 0.01）。综合试验结果,初步揭示了该短雄花序为一个赤霉素缺陷型突变体。