Construction and screening of suppression subtractive hybridization library of renal cell carcinoma

Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A+ RNA was isolated from RCC lines 786-O(tester) and renal cell(RC) lines HK-2 ( driver), respectiely. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit ( Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F’. All positive clones picked out were digested and some of which were sequenced. Results The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly,2 represented unknown genes and the other 48 derived from 36 known genes. Conclusion The quality of the SSH library of human RCC is reliable and is construction is the basis for further screening differentially expressed genes of RCC. 6 refs,4 figs, 1 tab.

Responses of Wheat (Triticum aestivum) to Grain Aphid (Sitobion avenae) Infestation and Mechanical Wounding Using a cDNA Subtractitve Library Approach

Aphids are major insect pests of cereal crops, acting as virus vectors as well as causing direct damage. The responses of commercial wheat (cv. Claire) to grain aphid (Sitobion avenae) infestation and mechanical wounding were investigated in this study, with the aim to eventually identify a source of molecular markers to breed wheat for enhanced insect resistance, and in particular for enhanced resistance to phloem-feeding insects. Mechanical wounding was included in this study as a comparison with aphid feeding to distinguish between insect-specific responses in wheat plants to those involved in a general wounding response. Wheat (Triticum spp.) is known to have partial resistance toward aphids [1]. The plant response and defence against insect feeding are complicated, but always follow the same principle: insect detection, signal transmission to initiate defence, changes in plant gene expression and subsequent production of defensive compounds, which may be targeted to the wound site to deter or kill insects. Defensive gene products/proteins reach the target area and deter or kill insects. Whether the last step is successful or not depends on the resistance and susceptibility of the plant towards that particular pest. In the light of this principle, it is important to detect changes in gene expression, first at the transcriptional level, which is useful for detection of early-stage responses, and then once sufficient time is allowed for the plant to produce defensive gene products, responses at the proteome level can be identified. Work presented in this study focuses on the changes at the transcriptional level;differential responses at the proteome level were investigated and presented in Ferry et al. 2011 [2] and Guan et al. 2015 [3]. Two cDNA subtractive hybridization libraries were constructed, one to identify transcripts involved in the responses to aphid infestation, and the second to identify transcripts involved in responses to mechanical wounding. Following subtractive hybridization, 520 and 800 clones were obtained from the subtractive hybridization between aphid-infested and un-infested wheat cDNAs and between mechanically wounded and un-wounded wheat cDNAs, respectively. Over 70% of the total clones were sequenced and 44% and 55% of sequenced clones were successfully identified by homology to known sequences held at NCBI with Blastx search engine in aphid-infested vs un-infested and mechanically wounded vs un-wounded cDNA subtractive libraries, respectively. These results reveal that the differences in the response of commercial wheat (cv. Claire) plants towards aphid infestation and mechanical wounding are subtle. Although the majority of differentially expressed putative genes after aphid infestation or mechanical wounding were involved in metabolic processes and photosynthesis, the majority of the genes expressed were different. Genes encoding glutathione transferase (GST), apoptosis and proteolysis were up-regulated after aphid feeding, suggesting their importance towards plant defence/tolerance against aphid attack. These results suggest that commercial wheat does have a certain degree of tolerance to aphids, but appears to lack a specific response to aphids;these findings are supported by those presented in Ferry et al. 2011 [2].

Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

Female inflorescence of Betula platyphylla was sampled at an interval of eachtwo days to analyze the background of gene expression in floral phase. On the basis of SMARTstrategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA wasfrom that of the others by RT-PCR which were subsequently used to construct a subtracted cDNAlibrary. The result of the ESTs (expression sequence tags) blastX showed that the genes in thesubtracted cDNA library could be mainly clustered into 5 groups related to metabolism,transportation and signal transduction, cell cycle, stress response, and regulation. Therelationship between gene expression and development was also discussed.

Construction of the Subtracted cDNA Library of Striatal Neurons Treated with Long-term Morphine

Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons （10^-5 mol/L for 72 hours）. To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein （CREB） mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons （62.85± 1.98） compared with normal striatal neurons （28.43 ± 1.46, P〈0.01）. Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma viral proto-oncogene 1 （Akt1 ; 122.10±2.17 vs 50.11±2.01, P〈0.01）, showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.

Expressed genes in regenerating rat liver after partial hepatectomy

AIM: To reveal the liver regeneration (LR) and its controlas well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers.METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of themshowed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressedin 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.

Analysis of Gene Expression Profile Induced by Water Stress in Upland Rice(Oryza sativa L.var.IRAT109)Seedlings using Subtractive Expressed Sequence Tags Library

To identify the water stress induced genes of upland rice cultivar IRAT109, which is resistant to drought, a subtractive cDNA library was developed from polyethylene glycol- （PEG） treated and non-treated seedlings by suppression subtractive hybridization, from which 2112 recombinant colonies were obtained, Eight hundred clones were selected randomly for sequencing analysis, and 384 unique expressed sequence tags （ESTs） were obtained, They were found to be involved in diverse biological processes, such as metabolism, transcription, signal transduction, protein synthesis and others, Notably a number of known functional genes in drought tolerance, including genes related to biosynthesis of osmoprotectants, defense against active oxygen, removal of toxic compounds, recovery of proteins and reinforcement of cell wall were also found in the study, Several genes related to deleterious responses were upregulated by PEG stress, The differential expression patterns of 11 SSH-derived ESTs were confirmed by real-time polymerase chain reaction.

Gene Expression Profiles of Response to Water Stress at the Jointing Stage in Wheat

Drought is one of the most adverse environmental factors that impact on plant growth and reduce crop yields. To decipher the molecular mechanism of drought tolerance, we constructed a cDNA library using suppression subtractive hybridization （SSH） and dissected the gene expression profiles in seedling and jointing wheat plants after stress with PEG-6000. A total of 2 046 ESTs from the jointing stage library （J-Lib） were allocated to 961 contigs. Among the ESTs, 265 uni-genes in 12 categories were identified on the basis of sequence similarities and functional classifications. Most were known to be involved in protection, directly or indirectly, against water stress. To determine differences in gene expression profiles for water stress responses at the jointing and seedling stages, data from the J-Lib were compared with those from a 2- leaf seedling library （S-Lib） constructed by the same method. Significant differences were observed between the two libraries for function-known genes; signal transduction genes were far more active at jointing than at the seedling stage.

Molecular Basis of Unique Branching Phenotypes in Salvia splendens and the Role of PSY

The branching system of higher plants plays a very important role in plant morphogenesis,and the number of branches can directly affect crop yield and the ornamental value of plants.It is a complicated development process involving complex molecular mechanisms.The‘Cailinghong’variety of Salvia splendens is characterized by its great branching ability with the ability to grow into a spherical form naturally,without pinching.To gain insight into the molecular events during the branching development of S.splendens,suppressive subtractive hybridization(SSH)technology was used to screen differentially expressed genes between the erect plant type(strain 35)and the spherical plant type(‘Cailinghong’).In total,96 and 116 unigenes were annotated.Four and eight unigenes up-regulated in‘Cailinghong’and strain 35,respectively,were associated with plant hormone anabolism and signal transduction,suggesting that they participate in the branching process.One of these genes,phytoene synthase(PSY),is a precursor of the new plant hormone group strigolactones.Using the PSY fragment(192 bp)as a template,the cDNA sequence of PSY in S.splendens was cloned and named SsPSY.A relative expression analysis and transgenic test results indicated that SsPSY plays an important role in lateral branch development in‘Cailinghong’.These results provide new insight into the molecular mechanisms underlying branching in S.splendens.

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma

Objective To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.Methods Polyadenylated RNA ［Poly (A)+ RNA］ was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors l and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.Results A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400?bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.Conclusions The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.

Identifcation and Characterization of 177 Unreported Genes Associated with Liver Regeneration

The mammalian liver has a very strong regeneration capacity after partial hepa- tectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression pro?les were studied by cluster and generalization analyses. Among them, 177 genes were identi?ed unre- ported and up- or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2–10 folds, and 16 genes were either up- or down-regulated at dif- ferent time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chro- mosomes. The cluster and generalization analyses showed that the gene expression pro?les are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same pro?les are similar, and those expressed in di?erent pro?les have less similarity. However, the types, characteristics and functions of the 177 genes remain to be further studied.