小麦与叶锈菌互作过程中LRRs类基因在转录水平的表达分析

本试验以小麦近等基因系TcLr26和Thatcher分别与叶锈菌生理小种260组成不亲和及亲和组合,基于RNA-Seq高通量测序数据库,通过基因表达差异分析,筛选了6个在接种后较0h表达明显变化的属于LRRs类基因的Unigenes,利用实时定量PCR(RT-qPCR)技术对这6个LRRs类基因在接种叶锈菌后的表达进行了分析,为深入研究这些LRRs类蛋白在小麦抗叶锈菌侵染过程中的作用机制奠定了重要试验基础。     

苦荞全生育期芦丁积累与其生物合成途径相关基因表达分析

【目的】探究苦荞全生育期芦丁含量变化与其合成途径关键酶基因和调控因子MYB基因表达量之间的相关性,以期进一步明确苦荞植株体内芦丁生物合成的分子机制。【方法】以九江苦荞为试验材料,整个生育期分别在萌发期、子叶期、真叶期、盛叶期、现蕾期、盛花期、灌浆期和籽粒成熟期共8个时期取材(S1—S8)。采用RT-PCR方法,克隆获得与黄酮类代谢相关的MYB类转录因子基因。采用T-coffee软件进行氨基酸同源序列比对及保守结构域分析。与拟南芥黄酮类代谢相关的MYB转录因子及荞麦同源MYB转录因子序列比对,基于邻近法构建系统进化树;实时荧光定量PCR技术分析芦丁合成途径中5个关键酶Ft CHS、Ft F3H、Ft4CL、Ft FLS-like和Ft UFGT及上述克隆到的MYB转录因子在不同组织中的表达模式。基于高效液相色谱法(HPLC)测定全生育期取材组织的芦丁含量。同时采用相关性分析方法分析不同组织中芦丁含量变化与基因表达模式的相关性。转换上述数据为矩阵,采用欧式距离法构建共表达层次聚类图谱。【结果】克隆到2个MYB类转录因子,即Ft MYB7和Ft MYB9,其核酸序列长度分别为876和912 bp,分别编码291和303个氨基酸残基。氨基酸序列同源性比对分析结果表明,二者均具有典型的R2R3保守结构域,为R2R3类型的MYB转录因子。结合Nr数据库中17个苦荞和3个拟南芥黄酮类代谢相关MYB转录因子同源氨基酸序列构建系统进化树,结果表明这22个基因分成6大类群,其中Ft MYB7属于第II类群,Ft MYB9属于第IV类群,二者分属于不同类群,暗示这2个MYB转录因子可能涉及调控植物生长发育过程中不同功能类型。荧光定量结果显示,Ft MYB7在S3(真叶期)和S5(现蕾期)基因相对表达量最高,达到501和867倍;Ft MYB9在S1(萌发期)和S2(子叶期)相对表达量最高,分别为34和72倍。上述基因表达量与芦丁含量变化模式相关性分析表明,8个生长时期中,Ft4CL、Ft CHS、Ft F3H、Ft UFGT和Ft MYB7相对表达模式与芦丁含量变化幅度呈正相关,其相关系数分别为0.748、0.683、0.704、0.890和0.862。而Ft FLS-like和Ft MYB9与芦丁含量的变化幅度呈负相关,其相关系数分别为-0.442和-0.501。【结论】Ft MYB7和Ft MYB9转录因子在整个生育时期中表达模式存在明显差异。其中Ft MYB7可能在苦荞芦丁积累的过程中起到正调控作用,而Ft MYB9则为负调控作用。     

甘蓝抗枯萎病基因FOC1植物表达载体构建及遗传转化

为研究甘蓝枯萎病抗性基因FOC1的抗性功能,利用前期克隆的FOC1基因,以pBI121质粒为植物表达载体,采用同源重组法构建FOC1基因的过表达载体;将构建好的重组质粒采用冻融法转入根癌农杆菌LBA4404菌株中,并通过农杆菌介导的甘蓝外植体转化法对感病甘蓝进行遗传转化,利用载体特异引物对获得的转基因植株进行PCR鉴定。结果表明,最终成功构建FOC1基因的过表达载体pBI121-35S-FOC1,并已成功整合到受体甘蓝基因组中。     

Arbuscular mycorrhizal fungi reduce ammonia emissions under different land-use types in agro-pastoral areas

Ammonia (NH₃) emissions, the most important nitrogen (N) loss form, always induce a series of environmental problems such as increased frequency of regional haze pollution, accelerated N deposition, and N eutrophication. Arbuscular mycorrhizal (AM) fungi play key roles in N cycling. However, it is still unclear whether AM fungi can alleviate N losses by reducing NH₃ emissions. The potential mechanisms by which AM fungi reduce NH₃ emissions in five land-use types (grazed grassland, mowed grassland, fenced grassland, artificial alfalfa grassland, and cropland) were explored in this study. Results showed that AM fungal inoculation significantly reduced NH₃ emissions, and the mycorrhizal responses of NH₃ emissions were determined by land-use type. Structural equation modeling (SEM) showed that AM fungi and land-use type directly affected NH₃ emissions. In addition, the reduction in NH₃ emissions was largely driven by the decline in soil NH⁺₄-N and pH and the increases in abundances of ammonia-oxidizing archaea (AOA) amoA and bacteria (AOB) amoB genes, urease activity, and plant N uptake induced by AM fungal inoculation and land-use type. The present results highlight that reducing the negative influence of agricultural intensification caused by land-use type changes on AM fungi should be considered to reduce N losses in agriculture and grassland ecosystems.     

植物病原细菌Pseudomonas syringae pv. tomato DC3000致病基因比较基因组及原核表达分析

番茄细菌性叶斑病是由Pseudomonas syringae pv. tomato引起的一种世界范围内广泛发生的植物细菌性病害,其模式菌株DC3000已完成全基因组测序。试验通过比较基因组学方法将该模式菌株基因组中的致病基因与其他已完成全基因组测序的植物病原细菌的基因组序列进行比对,得到3个DC3000菌株特有的致病基因PSPTO_4707、PSPTO_4708、PSPTO_4711,并对PSPTO_4711基因进行原核表达和蛋白活力检测,发现其表达的活性蛋白对拟南芥具有很强的毒性。     

高产纤维素酶绿色木霉基因工程菌的构建

[目的]构建CBHⅡ基因过量表达的绿色木霉工程菌。[方法]根据绿色木霉cDNA序列设计合成引物,PCR扩增CBHⅡ基因序列,将完整的目的基因与表达载体pCAMBIA1302连接,转入大肠杆菌DH5α中,获得重组质粒pCAMBIA1302-CBHⅠ。再将pCAM-BIA1302-CBHⅠ重组质粒与瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅱ（CBHⅡ）PcbhⅡ启动子片段连接,将潮霉素磷酸转移酶（hyg）基因片段插入PcbhⅡ启动子下游,转入大肠杆菌DH5α中,获得重组表达质粒pCAMBIA1302-CBHⅡ。[结果]构建的重组表达质粒电转化后经SDS-PAGE电泳检测,绿色木霉纤维素酶CBHII基因在原宿主菌中成功过量表达,证实CBHⅡ基因在PcbhⅡ启动子控制下进行高效表达。[结论]为高产纤维素酶系的工业化菌株构建奠定基础。     

Understanding the use of Bayes factor for testing candidate genes

After quantitative trait loci (QTL) detection, one of the main objectives of research is to identify the causal mutation explaining phenotypic differences. Candidate genes are usually selected according to the physiological mechanism of the trait and their location within the same region of the QTL. After detection of any polymorphism at the candidate gene sequence, it is important to determine whether the detected mutation is the one that causes the phenotypic variation. This is not, however, an easy task, because of the linkage disequilibrium between the genes located in the same region. Several methods have been proposed that consider the neutral marker information in validating the involvement of candidate genes. However, some statistical information may be lost because of the presence of both the QTL and candidate gene effects in the model of analysis. Here, the Bayes factor is suggested as an alternative and a procedure for its calculation between candidate gene and QTL models is presented. The procedure is illustrated with a simulation study and with an example consisting of three SNPs detected at the leptin receptor (LEPR) in an experimental intercross between Iberian and Landrace pigs. The results indicate that the Bayes factor procedure is more powerful than the classical approach.     

牛AMPK家族基因的研究进展

AMP激活的蛋白激酶（AMP-activated prorein kinase,AMPK）是一种能被腺苷一磷酸（AMP）激活的蛋白激酶。在动物应激（生理、营养、环境和疾病等）过程中起着重要作用。近年研究表明,AMPK在细胞和全身组织器官能量代谢平衡中起着举足轻重的作用,并且在调节物质代谢中起重要作用,其调节代谢过程主要包括葡萄糖转换,糖酵解和脂类代谢等。近年来在牛上已经进行了很多研究,其中包括其染色体定位及结构的确定等。本文将简要介绍AMPK的分子结构及功能,并对AMPK基因在牛上的研究进展进行综述。     

Functional and Structural Characterization of FAU Gene/Protein from Marine Sponge Suberites domuncula

Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with “higher” metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.     

高等植物适应盐逆境研究进展

土壤盐碱化已经成为制约农作物生长及产量的重要因子之一,寻求将盐碱化对植物的危害降低到最小程度的策略势在必行。关于植物对盐逆境适应能力的研究已成为全球关注的热点, 如何提高植物的耐盐能力也已成为研究的重中之重。深入探究高等植物适应盐逆境的机制,有助于提高植物耐盐性,增加作物产量和保护生态环境。本文就高等植物适应盐逆境的重点研究进展,综述了盐胁迫对植物的危害;植物耐盐的生理机制,包括渗透调节、营养元素平衡和增强抗氧化胁迫等;植物耐盐相关基因研究进展,包括离子转运蛋白基因、渗透调节相关基因、信号传导相关基因和细胞抗氧化相关基因等;提高植物耐盐性的途径。最后针对今后植物适应盐逆境方面的研究方向进行了展望。