日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

落叶松体细胞胚成熟阶段差异表达的基因及部分基因的表达谱分析

摘要: 为研究落叶松体细胞胚胎发生的分子机理, 文章以日本落叶松×华北落叶松杂种无性系胚性细胞系Y35体细胞胚成熟阶段培养物的cDNA为实验组, 继代培养阶段胚性愈伤组织的cDNA为对照组, 利用抑制性消减杂交技术(Suppression subtractive hybridization, SSH)构建了体细胞胚成熟阶段的差异表达基因文库。随机选取800个阳性克隆进行测序, 共获得468个UniGenes, 共将其分为19类, 功能分析结果表明: 这些UniGenes可能参与代谢、转录、信号转导、转运、细胞生长分裂、细胞结构、细胞命运、蛋白质合成与降解、防御等与个体发育密切相关的生物学过程。对部分ESTs的表达谱进行分析, 结果表明这些ESTs均在落叶松体细胞胚胎发生的不同阶段特异表达。     

Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes

Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82–89% similarity at the amino acid level to other F3H proteins. This is the first report on the gennomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.     

杨盘二孢菌诱导的杨树差异表达基因的功能分析和染色体定位

利用cDNA芯片技术从含有2,952个克隆的杨树芯片中筛选出1,160个受杨盘二孢菌诱导的基因。功能分析表明,该1,160个基因分别属于11个功能类别,除了功能未知基因外,参与新陈代谢、防御反应、信号传导及转录调控的基因最多,这4大类基因约占基因总数的42%。1,160个差异表达基因中有926个基因被定位于19条染色体上,其中被定位于第Ⅱ条染色体上的差异基因最多,共102个(11.0%),其次是第Ⅰ条染色体,共93个(10%),被定位到第ⅩⅦ条染色体上的差异基因最少,仅有11个,基因在染色体上的分布则表现为在部分染色体的末端区域存在大量的聚集,在中间区段则相对较少和排列稀疏,基因的这种分布情况与植物抗病的关系有待进一步研究。     

Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-κB p65 signal pathway

Tea polyphenols (TP) was investigated in rats for its protective effect on renal ischemia/reperfusion injury (RIRI). Rats were randomized into groups as follows: (I) sham group (n = 10); (II) RIRI group (n = 10); (III) RIRI + TP (100 mg/kg) group (n = 5); (IV) RIRI + TP (200 mg/kg) group (n = 5); (V) RIRI + TP+ Astragalus mongholicus aqueous extract (AMAE) (300 mg/kg + 100 mg/kg) group (n = 5). For the IRI + TP groups, rats were orally given with tea polyphenols (100, 200 and 300 mg/kg body weight) once daily 10 days before induction of ischemia, followed by renal IRI. For the sham group and RIRI group, rats were orally given with equal volume of saline once daily 10 days before induction of ischemia, followed by renal IRI. Results showed that tea polyphenol pretreatment significantly suppressed ROS level and MDA release. On the other hand, in rats subjected to ischemia–reperfusion, the activities of endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) showed recovery, whereas the levels of urea nitrogen and serum creatinine were reduced by administration of tea polyphenols orally for 10 days prior to ischemia–reperfusion. Moreover, tea polyphenol pretreatment significantly decreased TLR4 and NF-κB p65 protein expression levels in RIRI rats. At the same time, tea polyphenol pretreatment attenuated the increased level of serum IL-1β, IL-6, ICAM-1 and TNF-α, and enhanced IL-10 production in RIRI rats. Furthermore, tea polyphenol pretreatment significantly decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion, alleviating renal ischemia/reperfusion injury. These results cumulatively indicate that tea polyphenol pretreatment could suppress the TLR4/NF-κB p65 signaling pathway, protecting renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis, which implies that antioxidants may be a potential and effective agent for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TLR4/NF-κB p65 signal pathway. Moreover, supplement of AMAE can increased renal protection effect of TP.     

罗汉果果实不同发育时期SSH文库的构建

以罗汉果授粉后50 d和70 d果实为材料,利用抑制消减杂交技术构建了罗汉果果实在不同生育时期皂苷生物合成相关基因的差减cDNA文库.在正向差减文库(70 d为tester,50 d果实为driver)中随机挑选了641个cDNA阳性克隆测序,得到622条有效序列,重组率在96%以上.外源片段的长度分布在101～934...     

Identification of Genes Differentially Expressed Between Ochratoxin-Producing and Non-Producing Strains of Aspergillus westerdijkiae

Approximately 70 % of Aspergillus westerdijkiae strains are able to produce ochratoxin A (OTA), a nephrotoxic and carcinogenic mycotoxin which have been found in cereal and food commodities. Despite of its importance there is, up to now, no information available about which genes are differentially expressed between A. westerdijkiae ochratoxin-producing and non-producing strains. Using cDNA RDA approach we successfully sequenced 231 raw ESTs expected to be enriched in the ochratoxin-producing strain. BLASTX searches against the public databases showed that of these, 205 ESTs (79 %) exhibited significant similarities with proteins of known functions, 28 ESTs (11 %) had matches to hypothetical proteins, and the remaining 27 ESTs (10 %) had no significant hits. EST alignment resulted in a total of 14 non-redundant consensus sequences. Three putative genes encoding oxidoreductases were validated as up-expressed in the OTA producer strain using RT-qPCR approach. The expression of the putative genes encoding a cytochrome P450 family protein, 3-hydroxyphenylacetate-6-hydroxylase, and endoplasmic reticulum oxidoreductin were higher (32-, 2.8- and 20-fold respectively) in the OTA producer strain compared to the non-producer strain.     

Analysis of gene expression in non-regressed and regressed bovine corpus luteum tissue using a customized ovarian cDNA array

The lifespan of the bovine corpus luteum (CL) is an important factor in the control of normal ovarian cyclicity and the establishment and maintenance of pregnancy. There is increasing evidence that CL lifespan is regulated by alternative expression of genes that promote or inhibit luteolysis. To gain further insights into these events a 434 character ovarian cDNA array comprising genes attributed to key aspects of CL function including more than 100 anonymous expressed sequence tags (ESTs) was constructed and screened with alpha(33)P dATP labeled RNA isolated from non-regressed (n=6) and regressed (n=6) CL tissue. Significance analysis of microarrays (SAM) identified 15 genes that changed expression 1.7-fold or more with a false discovery rate of <5%. The differentially expressed genes encoded enzymes involved in steroid biosynthesis and oxygen radical metabolism and proteins involved in extracellular matrix remodeling, apoptosis and cell structure. Results for five of the differentially expressed genes including matrix gla protein and collagen alpha1(I) (extracellular matrix), glutathione-S-transferase alpha I (oxygen metabolism), clusterin (apoptosis) and scavenger receptor BI (steroid biosynthesis) were confirmed by Northern blot analysis and found to be significantly different (P<0.01) between non-regressed and regressed CL tissue. Collectively this study identified genes with recognized roles in CL regression, genes with potential roles in this process and genes whose function have yet to be defined in this event.