Identification and analysis of differentially expressed genes involved in dark-induced photoperiod response and senescence of soybean leaves by suppression subtractive hybridization

A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/16-h dark) and long-day(16-h light/8-h dark) conditions.A total of 148 clones were sequenced,representing 76 unique ESTs which corresponded to about 20% of 738 clones from the cDNA library and showed a significant up-regulation of at least three fold verified by dot blot hybridization.The putative functions of ESTs were predicted by Blastn and Blastx.The 43 differentially expressed genes identified by subtractions were classified according to their putative functions generated by Blast analysis.Genetic functional analysis indicated that putative proteins encoded by these genes were related to diverse functions during organism development,which include biological regulation pathways such as transcription,signal transduction and programmed cell death,protein,nucleic acid and carbohydrate macromolecule degradation,the cell wall modification,primary and secondary metabolism and stress response.Two soybean transcription factors enhanced in SD conditions,GAMYB-binding protein and DNA binding protein RAV cDNAs that may be involved in SD soybean photoperiod response,had been isolated using 5'-and 3'-rapid amplification of cDNA ends(RACE)(Genbank Accession numbers DQ112540 and DQ147914).

Differential Regulation of Proteins and a Possible Role for Manganese Superoxide Dismutase in Bioluminescence of Panellus stipticus Revealed by Suppression Subtractive Hybridization

Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.

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.

Identification of cadmium-induced genes in maize seedlings by suppression subtractive hybridization

A maize variety,Huatian-1,had an unusuallylow translocation rate of cadmium(Cd)(59.6 mg·kg^(-1)inthe roots and 0.093 mg·kg^(-1)in the grain)compared to24 other varieties while being grown in soils with16.50 mg·kg^(-1)Cd.This indicates that this particularspecies may have special mechanisms that affect theabsorption and translocation pattern of Cd.In this paper,the technique of suppression subtractive hybridization(SSH)was used to isolate and identify Cd-induced genesfrom Huatian-1 hydroponically exposed to 0.1mMCdCl_(2) for 1 h,12 h,24 h,and 48 h.We found a total of15 differentially expressed genes in the four groups;2,3,4,and 6 genes were from the groups of 1 h,12 h,24 h,and48 h treatment,respectively.Phospholipase PLDb1mRNA,adenosine triphosphate(ATP)phosphoribosyltransferase 2,and Sp17 were turned on in the maize inresponse to Cd stress,and it might provide new clues toexplain the mechanism of maize tolerance to Cd.

Identifcation 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 88genes. 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 werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Identification and Expression Analysis of Salinity-induced Genes in Rangpur lime(Citrus limonia)

Among the economically important horticultural crops,citrus is one of the most vulnerable crops to soil salinity.Rangpur lime is more tolerant to high soil salinity than other commonly used citrus rootstocks.However,the molecular mechanism involved in salinity tolerance has not been explored in Rangpur lime.In this study,a cDNA library was constructed from leaves of Rangpur lime watered for 30 days with 100mmol·L^−1 NaCl in tap water or tap water using suppression subtractive hybridization(SSH).Two hundred cDNA clones randomly selected from this library were sequenced,and an average of 569 bp was obtained from the majority of clones.Fifty-six salinity-induced genes,showing 2-to 6-fold increases in their expression levels,were identified by macroarray hybridizations.Salinity-induced genes were associated with transcription(5.36%),stress response and signaling(21.43%),metabolism(16.07%),transport facilitation(10.71%),photosynthesis(10.71%),protein synthesis and fate(19.64%)and cellular biogenesis(3.57%).Stress response-and signaling-related genes constituted the largest functional group,associated with the production of compatible solutes,regulation of stomatal movement,lipid modification,oxidative stress,antioxidant defense,and stress signaling.Expression levels of 13 identified genes were induced 1.8-to 3.1-fold,which were validated in salt-treated and untreated Rangpur lime.The functions of salinity-induced,stress-related genes and their potential roles in salinity tolerance in Rangpur lime were discussed.

Identification of differentially expressed genes in two new human bladder carcinoma cell lines

Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

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.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio)by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthelowed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of Antranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes).RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary,spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart,kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtelIogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.