PCR-select differential screening of a subtracted cDNA library of heat-adapted rat liver

AIM: To reveal the molecular mechanisms of heat adaptation by study the change of gene expression in rat liver when the rats exposed to hyperthermia stress and heat adaptation. METHODS: Differentially expressed genes in rat liver in association with heat adaptation were identified using the suppression subtractive hybridization (SSH) technique to prepare subtracted cDNA libraries in heat adaptation. The PCR-amplified cDNA fragments generated by SSH were then ligated into the plasmid pGEM-T (Promega). PCR-select differential screening technique was used to filtrate differentially expressed genes. Each of these differentially expressed genes was sequenced and was compared to known sequences in the GenBank database. RESULTS: eight up-regulating expressed genes segments were isolated from the up-regulating differentially expressed library, three of which were known gene such as p53 and five genes were new expressed sequence tags. CONCLUSION: Up-regulating expressed genes such as p53 maybe participate in the signal transduction pathway of heat adaptation.     

Construction of lymphocyte subtracted cDNA library of unstable angina

AIM:To construct a subtracted cDNA library of differentially expressed genes in human unstable angina lymphocytes.METHODS:Suppression subtractive hybridizations (SSH) were performed between the patients with unstable angina pectoris and stable angina pectoris. Lymphocyte RNA, the obtained forward and reverse cDNA fragments were directly inserted into T/A cloning vector and transformed into E.coli JM 109 to construct a subtractive cDNA library. The inserting fragments were screened by blue and while blot screening and bacterium liqulid PCR.RESULTS:Each subtractive cDNA library contained more than 2000 positive bacteria clones. Most of them distributed between 200-600 bp inserts.CONCLUSION:The library is efficient and lays solid foundation for screening and cloning new and specific expressed genes in unstable angina lymphocyte RNA.     

Identification of differentially expressed genes associated with cell adhesion and immune regulation in peripheral blood eosinophils from asthmatic patients with suppression subtractive hybridization

AIM:To determine differentially expressed genes associated with cell adhesion and immune regulation in peripheral blood eosinophils from asthmatic patients. METHODS:Peripheral blood eosinophils were isolated from the asthmatic patients at the time of exacerbation and after improvement. Total RNA was extracted. Super SMART PCR cDNA was synthesized, suppression subtractive hybridization (SSH) and PCR-select differential screening technology were used to detect expressed genes. The differentially expressed genes were sequenced. RESULTS:High efficiency subtractive cDNA library was constructed successfully. Differential screening identified 15 differentially expressed genes, which were Charcot-Leyden crystal protein (CLC protein;galectin-10), putative pre-mRNA splicing regulator female-lethal (2D), aquaporin 9 (AQP9), IL-8, slingshot 2L (SSH-2L), PP1 catalytic subunit, beta isoform, helicase with zinc finger domain (HELZ), β2-microglobin (β2-MG) and a gene associated with mitochondrion. CONCLUSION:Increased expression of these genes might be associated with eosinophil migration, adhesion and immune regulation. Intervention of these pathways may provide a theoretical base for future new targeting treatment for asthma.     

应用抑制性消减杂交分离板栗短雄花序芽变相关基因

利用抑制性消减杂交技术分离了板栗正常花序与芽变花序之间表达的差异cDNA片段。以芽变板栗雄花序cDNA为试验方(tester),以对照板栗雄花序cDNA为驱动方(driver),构建了一个板栗芽变花序消减文库。菌落PCR显示插入片段主要在100～1000 bp,文库质量良好。通过对文库阳性克隆随机测序共得到了30个有效EST序列。利用Blast同源性比较,其中一些EST可能与板栗雄花序发育中变短的生物学形态形成密切相关,为后续板栗短雄花序芽变相关基因的研究奠定了基础。     

荔枝采后果皮褐变过程中差异表达基因的SSH分析

 以‘妃子笑’荔枝果实为材料,采用抑制差减杂交（SSH）与cDNA 微阵列技术相结合,研 究了采后荔枝果皮褐变过程中的基因差异表达。分别以采后0 h 与32 h 的果皮总RNA 为驱动组与检测组, 构建了正向与反向SSH 文库,分别获得了282 个与76 个阳性克隆。通过cDNA 微阵列杂交筛选获得了在 采后32 h 果皮中上调表达克隆17 个,下调表达克隆49 个,分别代表了在采后32 h 果皮中上调表达基因 16 个和下调表达基因17 个,其中有较多的热击蛋白基因、糖代谢相关基因、细胞壁代谢相关基因等。 RT-PCR 检测基因表达结果与cDNA 微阵列杂交结果一致。     

抑制性消减杂交分离苹果缺铁诱导的相关基因

以铁高效基因型植物———苹果属小金海棠(Malus xiaojinensis Cheng et J iang) 为材料, 利用抑制性消减杂交( SSH) 方法, 成功构建了小金海棠缺铁条件下的消减cDNA文库。该文库包含1 152个克隆, 通过PCR检测插入片段大小在300～1 000 bp之间。对构建的消减cDNA文库进行差异筛选, 得到差异表达片段( ESTs) 105个。GenBank上BLAST结果如下: 50个ESTs与其它物种同源性较高, 视为已知基因, 并根据其功能分为9个组; 32个ESTs在GenBank中无法查到对应的同源序列, 可能代表了新基因;另外23个是与已知ESTs重复的基因片段。     

茄子单性结实候选基因的表达分析

对茄子单性结实抑制差减文库中的1 248个差异表达unigenes在GeneBank数据库中进行BLAST比对分析,结果表明1 109个unigenes具有同源序列,其编码的蛋白包括甲硫氨酸亚砜还原酶、MADS-box转录因子、组蛋白和隐花色素等,涉及到果实发育的多个方面,139个unigenes没有同源序列,可能为新基因。利用荧光定量PCR研究10个差异表达unigenes在茄子单性结实果实和非单性结实果实发育过程中的表达模式,分析表明,在低温条件下,unigenes Z569和Z707在单性结实果实发育中特异表达,可能与单性结实果实的形成有关,可作为研究单性结实的候选基因。     

黄瓜霜霉病抗性相关基因的初步研究

 以黄瓜高抗霜霉病自交系为材料,构建了两个抑制差减文库,经反向Northern杂交筛选、测序和序列比对,筛选到3个未知功能抗病相关基因。荧光实时定量RT-PCR分析结果表明,未知功能抗病相关基因2I15（GD254229）、3C19（GD254243）和1O08（GD254207）在抗病自交系接种霜霉病病原菌后,可早期高丰度特异上调表达,而在感病自交系中特异上调表达丰度较低或被特异抑制表达。水杨酸（SA）可诱导1O08特异上调表达,抑制2I15和3C19的表达;茉莉酸（JA）处理可诱导1O08特异上调表达,而抑制2I15的表达。ABA处理可显著诱导3C19和1O08特异上调表达,而抑制2I15的表达。机械伤害、高盐、冷害和热激等其它非生物胁迫可显著抑制或诱导这些基因的表达,因此推测,所筛选到的功能未知基因可能具有参与生物和非生物胁迫反应的作用     

Screening and identification of differentially expressed genes in hippocampus of mice during hypobaric hypoxic delayed preconditioning

AIM: This study was designed to explore the differentially expressed genes between hypobaric hypoxic delayed preconditioning (HHDP) and normal mouse hippocampus. METHODS: HHDP was produced by treating the animals at a 7 000 m high altitude for 2.5 h/d for 3 d. At 36 h after last time decompression, total RNA was isolated from hippocampus. cDNA was synthesized and amplified by SMART PCR. cDNA libraries of differentially expressed gene between HHDP and control hippocampus were constructed. 452 clones from forward (subtracted control from preconditioning) cDNA library and 74 clones from backward (subtracted preconditioning from control) one were screened by reverse Northern hybridization. RESULTS: Screening with subtracted probes, hybridization signal of 85 gene fractions decreased and that of 217 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Screening with unsubtracted probe, hybridization signal of 44 gene fractions decreased and that of 135 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Some of the clones had been sequenced. Analysis and comparison with the data of GenBank were performed. The results showed that mouse cytochrome C oxidase subunit 1, NADH dehydrogenase subunit 1 and 6, deleted in split-hand/split-foot 1 region (DSS1) and cDNA corresponding to clone IMAGE: 5251089 of mice cDNA library were increased in hippocampus of HHDP mice. cDNA corresponding to clone IMAGE: 3593193, mus musculus adult male olfactory brain cDNA and mus musculus bladder RCB-0544 MBT-2 cDAN were decreased in hippocampus of HHDP mice. CONCLUSION: Many genes expresses differentially in hippocampus of mice during HHDP. This may be one of the molecular mechanisms of HHDP.     

Construction of atherosclerosis full-length fully human antibody libraries by mammalian cell surface display technique

AIM: To construct atherosclerosis (As) full-length fully human antibody libraries by mammalian cell surface display technique for looking for the atherosclerotic therapeutic antibody. METHODS: The vector pcDNA-DHL was constructed by remounding the vector pcDNA5/FRT. The human total RNA from human peripheral blood mononuclear cells was isolated, and then reverse transcripation of RNA to cDNA was performed. The genes encoding the heavy chain variable regions and kappa light chains of the antibodies were amplified by PCR, and the product of PCR was connected to the vector pcDNA-DHL and the vector pDHL-As. The library DNAs were transfected into Flp-In^TM-CHO (FCHO) cells, the expression of the full-length human antibodies on the surface of cells was analyzed by flow cytometry. The cells that were successfully transfected with pDHL-As were screened with hygromycin, and then the full-length antibody library of atherosclerosis was obtained. RESULTS: The vector pcDNA-DHL were constructed successfully. The heavy chain gene library constructed showed a diversity of 1.79×10⁵, and the kappa light chain gene library had a diversity of 1.80×10⁵. Theoretically, the diversity of antibody library is 2.32×10¹⁰. The antibody library of atherosclerosis pDHL-As expressed on the surface of Flp-In^TM-CHO cells. CONCLUSION: The full-length human antibody cell library of atherosclerosis was constructed successfully, and 45 FCHO cells that expressing anti-oxidized low density lipoprotein antibodies were selected.     

Identification of some expressed sequence tags in cerebellum of rat

AIM: To obtain differentially expressed cDNA fragments in the cerebellum of rats and screen unknown expressed sequence tag (EST). METHODS:Suppression subtractive hybridization (SSH) was carried out, in which the cDNA fragments of cerebellum were taken as "tester" and correspondingly that of cerebrum and brain stem as "driver". The homogeneous sequences between the tester and driver were excluded and the rare sequences in cerebellum were enriched by SSH. The differentially expressed cDNA fragments were further cloned for the construction of subtracted cDNA libraries and sequencing. RESULTS: 32 clones were selected and 34 cDNA fragments were sequenced. 8 of 32 were proved to be true positive with reverse Northern assay, 13 of 34 fragments were identified to be new cDNA fragment and given the gene sequence numbers by GenBank (AW288461-AW288474).CONCLUSION:SSH is very useful method for screening differentially expressed genes.Our data may be helpful to understand the molecular mechanism of brain function.     

Isolation and expression analysis of differentially expressed genes in stem tissue of the Greek lemon cv. Adamopoulou

Lemon (Citrus limon (L.) Burm. f.) is susceptible to mal secco, a serious vascular disease caused by the fungus Phoma tracheiphila (Petri) Kant. and Gik., as well as low temperatures. The greek lemon cultivar Adamopoulou, thought to be derived from the Portuguese cultivar Lisbon, exhibits enhanced resistance to mal secco and cold as opposed to cv. Lisbon. Suppression subtractive hybridization (SSH) was employed for the isolation of differentially expressed genes in lemon stem tissue. A subtractive cDNA library was constructed and a total of 296 clones were sequenced. The obtained sequences were edited, resulting in 56 non-redundant ESTs. Sequence analysis revealed homology to previously identified genes involved in defense mechanisms against biotic and abiotic stresses, as well as sequences with no significant similarity in the GenBank. Selected ESTs were analyzed by real-time PCR for confirmation purposes. This analysis revealed significant expression differences between the two cultivars for genes expressing allantoinase, ultraviolet-B-repressible protein, 4-coumarate:CoA ligase and other proteins that are known to be upregulated under biotic and abiotic stress conditions.     

琯溪蜜柚及其早熟红肉突变体成熟果实SSH 文库的构建及初步分析

 分别以琯溪蜜柚及其突变体红肉蜜柚（成熟期早20 ~ 25 d）的成熟果肉cDNA 作驱赶子和检测子,利用结合分子镜像选择技术的SSH 技术,构建了红肉蜜柚和琯溪蜜柚的正向差减cDNA 文库。通过PCR 检测cDNA 克隆外源插入片段,其大小介于100 ~ 1 000 bp。通过点杂交差异筛选文库,得到102 个表达增强2 倍或以上的克隆并测序,结果表明这102 个克隆代表44 个Unigenes,通过序列同源性比对分析,发现获得的Unigenes 在功能上主要涉及信号传导、蛋白质合成、应激反应、转运等代谢反应。     

Identification of differentially expressed genes during flower opening by suppression subtractive hybridization and cDNA microarray analysis in Eustoma grandiflorum

The forward and reverse suppression subtractive cDNA libraries were constructed in petals of Eustoma grandiflorum at bud stage (stage 1) and anthesis (stage 7). Approximately 1000 clones were isolated from stage 1- (S1) and stage 7-specific (S7) libraries. The clones were sequenced and assembled, which yielded 98 contigs and 444 singletons. BLAST search was conducted on these assembled sequences. Generally, probes isolated from the S7 library exhibited higher expression at stage 7 by microarray analysis, as did those of the S1 library at stage 1. A clone set from the S7 library contained genes from later steps of anthocyanin biosynthesis pathway, terpene synthases, GAST (gibberellic acid-stimulated) family proteins, xyloglucan endotransglucosylase/hydrolase, glycosidases, and stress- and senescence-related proteins. In contrast, the S1 library contained genes associated with flavonol biosynthesis, phenylpropanoid metabolism, terpenoid metabolism, and floral organ development. Gene expression profiling for flavonoid biosynthesis was in accordance with preferential accumulation of flavonols at bud stages and anthocyanins at anthesis.     

利用SSH分离TDZ诱导金钗石斛成花相关基因

 以金钗石斛（Dendrobium nobile Lindl.）腋芽为材料,利用抑制消减杂交（SSH）技术构建了TDZ处理后金钗石斛腋芽的正向和反向SSH文库。通过PCR和反向Northern杂交验证后,得到的正向文库的314个阳性克隆和反向文库的59个克隆测序后经过聚类,最终得到39条受TDZ正向调控的EST序列,以及21条受其反向调控的EST序列。对其中某些差异基因进行半定量RT-PCR,结果显示这些基因的表达受TDZ的影响。利用Blastn和Blastx进行比对分析,其中20个没有同源序列,属于未知功能基因。其余序列的同源基因编码的产物参与包括光合作用、合成代谢、转录调控等多种生物学过程。在反向文库中分离到1个MADS-box基因的同源序列和1个Sos同源基因,而正向文库中存在多个反转录转座子,这些结果表明TDZ可以影响开花过程中的某些转录因子和信号基因的表达,进而促进金钗石斛开花。