抑制差减杂交分离赤霉病菌诱导的小麦特异表达基因

为了全面探索小麦赤霉病抗性机理,以抗赤霉病小麦品种‘苏麦3号’及其感病的近等基因系(Hwo4)为实验材料,构建了一个‘苏麦3号’受赤霉病菌诱导表达的正向差减杂交文库。随机选取了141个阳性克隆测序,共获得133条通读EST。将获得的EST去除载体及接头序列后,利用CAP3软件进行聚类分析,133条EST被聚成90个序列重叠群(contigs),片段大小介于106~643 bp间,平均长度为274 bp。利用NCBI的Blastx软件对序列进行蛋白序列同源性比对,结果显示76条序列在蛋白数据库中可以找到同源序列,功能涉及能量代谢、物质代谢、疾病/防御、转录及细胞结构等。其中以能量及物质代谢相关的基因数量最多,分别占总EST的21.1%及17.1%;其次是与抗病及防御反应有关的EST,数量占总EST的15.8%。进一步的分析表明与抗病及防御相关的EST功能主要涉及抗氧化、细胞解毒及相关物质的代谢。     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理(submergence-treated, ST)的玉米(Zea mays L.)幼苗根部cDNA为目标群体,未处理(untreated, UT)的玉米幼苗根部cDNA为对照群体,进行抑制差减杂交.用经过UT差减的ST cDNA构建了一个含有大约2 000个独立克隆的差减文库.对随机挑取的408个克隆进行差异筛选,获得了184个在ST中特异表达或表达增强的候选克隆.对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段.GenBank中BLAST查询结果表明:6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%～90%;21个克隆在GenBank中无法查到对应的同源序列,可能代表了新基因,或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性.     

人参植物皂苷生物合成相关新基因的筛选与鉴定

人参植物根进行的特定发育过程在药用次生物———人参皂苷生物合成和累积中发挥重要作用。为从人参根中分离出人参皂苷生物合成相关基因 ,采用抑制差减杂交技术 ,构建四年和一年生人参根组织mRNA群体间正向差减cDNA文库。对从差减文库中筛选的 4 0个阳性cDNA克隆进行酶切、PCR与逆向Northern斑点杂交鉴定、DNA测序以及核苷酸序列同源性比较。结果表明 ,获得的 6个差减克隆在GenBank/DDBJ/BMBL无对应的同源基因 ,代表新基因序列。与此同时 ,使用Northern印迹杂交验证及半定量RT PCR进一步确认 ,6个转录本为根发育阶段差异性表达基因。因而提示 ,它们可能在人参皂苷生物合成中发挥了重要作用。此外 ,在人参茎、叶与种子中亦能检测到上述基因转录本的表达。目前 ,6个新基因已被命名 ,在GenBank注册并获登录号 ,为克隆上述新基因cDNA全长序列及深入鉴定其在人参皂苷生物合成中的功能提供了重要实验依据。     

人血小板生成素cDNA转导抑制小鼠移植肉瘤(S180)的生长

以小鼠肉瘤S180为模型 ,研究人血小板生成素 (TPO)在肿瘤基因治疗方面的价值 .在小鼠接种瘤细胞前 2周 ,或在接种肿瘤细胞同时 ,给小鼠注射 10 0 μgTPO表达质粒 (pcDNA3 hTPO) ,在 2周和 4周时肿瘤平均重量明显小于单纯质粒 (pcDNA3)注射和生理盐水对照组 (提前转导组P <0 0 5 ,同时转导组P >0 0 5 ) .当肿瘤移植量为 1× 10 6时 ,2周时肿瘤的重量依次分别为 1 75 6g、3 6 37g和 3 92 6g ;当肿瘤移植量为 1× 10 5,2周时肿瘤的重量依次分别为 0 5 76g、5 6 1g和 5 84g .在转导基因 2周时 ,移植 1× 10 5肿瘤细胞组有 4 10受试鼠完全未形成肿瘤或肿瘤的生长被中止在早期阶段 .流式细胞分析发现 ,TPO转导后 ,肉瘤内浸润淋巴细胞从以CD8+为主转变为以CD4 +或CD4 +CD8+为主 .不过 ,TPO转导对瘤细胞在体外的增殖无明显改变 .TPO转导鼠的血清对S180细胞在体外的生长速度也无影响 .推测T淋巴细胞参与了TPO基因转导所产生的抗肿瘤作用     

应用抑制消减杂交和cDNA芯片技术筛选流行性感冒病毒感染相关基因

病毒基因组有限的编码能力和以病毒蛋白为靶的抗病毒药物易出现耐药性,使从病毒感染宿主筛选病毒感染相关生物大分子作为抗病毒药靶和诊断标志物成为新的研究方向。为了筛选流行性感冒（流感）病毒感染相关基因,采用抑制消减杂交（suppression subtractive hybridization,SSH)技术,以流感病毒A／鲁防／93－9（H3N2）感染的MDCK细胞及正常MDCK细胞为材料,构建病毒感染特异性差减cDNA文库。从文库中随机挑取约800个克隆,PCR扩增其中插入片段,经纯化、紫外定量后,用基因芯片自动点样仪点在氨基片上,制备cDNA芯片。将流感病毒感染的MDCK细胞和正常MDCK细胞的总RNA分别用Cy3、Cy5反转录荧光标记后,与cDNA芯片杂交,用芯片扫描仪扫描获得芯片杂交信号,经阳性对照校正和归一化处理后,以如下条件作为判定基因差异表达的标准;（a)Cy3与Cy5的信号比值大于1．5（正常细胞用Cy5标记）或小于0．67（正常细胞用Cy3标记）;（b)Cy3和Cy5信号值之一必须大于1000。经cDNA芯片筛选获得了18个流感病毒感染特异性克隆,经测序和生物信息学分析发现均为流感病毒感染相关新基因EST。流感病毒感染相关基因cDNA片段的获得,为新型病毒药靶诊断标志物发现和功能研究提供了基础。     

Gene expression profile favoring phenotypic reversion: a clue for mechanism of tumor suppression by NF-IL6 3'UTR

Transfection of cDNA in 3'untranslated region of human nuclear factor for interleukin-6 (NF-IL6 3'UTR) induced tumor suppression in a human hepatoma cell line. cDNA array analysis was used to reveal changes in gene expression profile leading to tumor suppression The results indicate that this suppression was not due to activation of dsRNA-dependent protein kinase, nor to inactivation of oncogenes; rather, all the changes in expression of known genes, induced by NF-IL6 3'UTR cDNA may be ascribed to the suppression of cellular malignancy. Therefore, our results imply that this 3'untranslated region may have played role of a regulator of gene expression profile.     

Identification of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88 genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes were firstly associated with UL. Three genes with notable difference were selected for Northern confirmation. Our results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate, testis, liver, heart and skeletal muscle.     

抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。     

Does allelopathy offer real promise for practical weed management and for explaining rhizosphere interactions involving higher plants?

This paper selectively reviews developments in the study of allelopathy, principally from a chemical standpoint. Particular attention is given to recently discovered aspects of signalling within the rhizosphere rather than to direct physiological effects caused by broadly active allelopathic agents with only a brief review of the role of bulk generation of weakly biocidal material. Many of the chemicals responsible for allelopathic effects have been identified and the biosynthesis and modes of action of a number are included. The paper also highlights instances where signalling aspects of allelopathy may be exploited for practical weed control purposes, and how the study of allelopathic components of the rhizosphere metabolome might be studied more directly by means of plant genomics.     

Bacterial cupredoxin azurin hijacks cellular signaling networks: Protein–protein interactions and cancer therapy

Azurin secreted by Pseudomonas aeruginosa is an anticancer bacteriocin, which preferentially enters human cancer cells and induces apoptosis or growth inhibition. It turns out that azurin is a multi‐target anticancer agent interfering in the p53 signaling pathway and the non‐receptor tyrosine kinases signaling pathway. This suggests that azurin exerts its anticancer activity by interacting with multiple targets and interfering in multiple steps in disease progression. Therefore, azurin could overcome resistance to therapy. Besides azurin, putative bacteriocins that possess functional properties similar to those of azurin have been identified in more bacteria species. A systematic investigation on the anticancer mechanisms of azurin and the azurin‐like bacteriocins will provide more and better options in cancer therapy. In this review, we summarize how azurin and the derived peptides hijack key cellular regulators or cell surface receptors to remodel the cellular signaling networks. In particular, we highlight the necessity of determining the structure of azurin/p53 complex and investigating the influence of post‐translational modifications on interactions between azurin and p53. Therapeutic applications of azurin and derived peptides are also discussed.