Suppressive subtractive hybridization of and differences in gene expression content of calcifying and noncalcifying cultures of Emiliania huxleyi strain 1516

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

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．     

Screening and comparison of differentially expressed genes between one MDR-TB strain and the virulent M. tuberculosis H37Rv

To screen and compare the differentially expressed genes between one MDR-TB strain separated from one child patient and the virulent Mycobacterium tuberculosis H37Rv, suppression subtractive hybridization (SSH) technology was used to build a library of cDNAs that were differentially expressed in the MDR and H37Rv. From this cDNA library, genes that were expressed in the MDR-TB but not in the H37Rv were selected for gene sequencing and homology analysis; 113 positive clones were obtained, their cDNA fragments were sequenced, and homology analysis was performed. Four novel sequences were identified. The results provide a partial list of genes differentially expressed in MDR-TB and four novel genes were found. Identification of these genes may contribute to our understanding of MDR-TB development.     

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis. Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

Isolation of cold stress-responsive genes from Lepidium latifolium by suppressive subtraction hybridization

Suppression subtraction hybridization (SSH) libraries were constructed from RNA isolated from leaves of control and cold stress-induced Lepidium latifolium, a cold-tolerant plant species from high altitudes for isolation of cold-responsive genes. A total of 500 clones were obtained from the cold stress library. Dot blot expression analysis identified 157 clones that were upregulated and 75 that were downregulated during cold stress. These clones selected on the basis of their expression patterns on dot blot were sequenced. As much as 27 and 17 genes were identified from the forward and reverse libraries, respectively. The genes identified revealed homology with genes involved in diverse processes such as gene regulation/signaling, photosynthesis, DNA damage repair protein, pathogenesis-related protein, senescence-associated proteins and proteins with unknown functions.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

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。     

植入前期与分娩前期子宫差别表达基因筛选及表达

采用抑制消减杂交技术 (suppression subtractive hybridization , SSH) ,以 SD 大鼠为实验材料,选取妊娠过程中植入前期 ( 第 5 天, D5) 和分娩前期 ( 第 19 天, D19) 子宫分别作为驱动方 (driver) 和实验方 (tester) ,进行抑制消减杂交,获得的消减文库经差异筛选得到 70 个阳性克隆 . 序列测定和同源对比分析表明,这些克隆所代表的基因在大鼠基因库中分别与 8 个已知基因有 90 ％～ 100 ％不等的同源性 . 这些基因均差别表达于分娩前期子宫组织中,其中首次发现尿鸟苷蛋白和干扰素诱导蛋白 16 在 SD 大鼠妊娠子宫中有表达 . RT-PCR 及半定量分析显示,尿鸟苷蛋白基因在妊娠第 19 天子宫中的表达显著高于妊娠第 5 天 (P < 0.001) ,而干扰素诱导蛋白 16 差异表达不明显 . 在妊娠 D6 、 D9 和 D12 的子宫中尿鸟苷蛋白基因自胚泡植入后其表达逐渐上升,妊娠 D15 下降,在妊娠 D19 表达量最高 . 结果提示特异表达的尿鸟苷蛋白基因可能与分娩有关 .