Isolation of the drought up-regulated gene in Alternanthera philoxeroides by differential display technique

Under drought stress, adventitious roots of Alternanthera philoxeroides seedlings will grow long thick fleshy roots, which are assumed to improve performance of the plant by more efficient reservation and absorption of water from deep soil layers. In this study, the differential display technique was used to clone morphogenesis-related genes from A. philoxeroides roots treated with drought, which would help to improve crop plants＇ drought-tolerance by transgenic method; by 15 pairs of primer combinations, twenty putative drought up-regulated gene segments induced by drought were obtained; and one of them was confirmed by reverse northern blot, and subsequently cloned and sequenced. A homologous analysis revealed that it might be a new sequence. Semi-quantitative RT-PCR analysis showed that the gene was up-regulated by drought and salt stress.

Application of mRNA Differential Display to the Identification of Genes Related to Embryonic Development

mRNA differential display was established by Liang and Pardee in 1992 for the purpose of displaying the mRNA differences between two tissues. The early embryonic development in animals is primarily controlled by the maternal RNAs stored in egg. These mRNAs are being degraded as the development proceeds. In some animals, such as fish and amphibian, new transcripts do not appear until the midblastula stage (midblastula transition, MBT). In other animals, for example in mouse, the zygotic genes are expressed during very early stages of development. The mRNA programmed synthesis and degradation during embryonic development controls the cell differentiation, germlayer formation and pattern formation. All these mRNA changes could be displayed side by side as cDNA band differences by mRNA differential display and the genes corresponding to these differential mRNAs could thus be obtained.

Progress in mRNA Differential Display

The mRNA Differential Display is a new molecular biological strategy for detecting and characterizing altered gene expression in eukaryotic cells which wad developed in 1992.Because of its simplicity,sensitivity and reproducibility,this method should find wide-ranging underpaid application developmental and molecular biology.Therecent successful applications of this method to gene hunting and the technological improvement promise great potential of mRNA Differential Display.

ESTABLISHMENT OF DIFFERENTIAL DISPLAY mRNA AND ITS APPLICATION IN THE STUDY ON THE MECHANISM OF LUNG CANCER INDUCED BY RADIATION

Objective: A method for separating mRNAs by means of the polymerase chain reaction (differential display mRNA), and identifying the genes related to radiation-induced lung cancer was introduced. Methods: The RNAs were isolated from two pairs of samples, SV40-immortalized human fetal tracheal fibroblast cell (SHTF) versus αSHTF cell (transformed SHTF cell induced by α particles) and lung cancer tissue versus normal lung tissue obtained from one miner, and amplified by RTPCR. The differential expressed gene fragments were displayed by autoradiograph or silver nitrate stain. Results: The differential display mRNA method was established using both cell and tissue samples. The bands stained by silver nitrate were clearer than those on X-ray film. The rate of reamplification of differentially expressed gene fragments stained by silver nitrate is 80%, higher than that by autoradiograph, 50%. Conclusion: Differential display mRNA method was established successfully on both cell and tissue samples. The modified method for staining band increased the rate of reamplification and established the basis for confirming relative genes.

Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display

RNAs isolated from ammonium- and nitrate-treated rice leaves were used to screen differentially expressed genes through mRNA differential display. A total of 72 bands appeared significant differences and some of them were further confirmed by reverse Northern and Northern blot. The results showed that two genes, A-02 （Oryza sativa drought stress related mRNA） and A-03 （Zea mays partial mRNA for TFIIB-related protein） were highly up-regulated in the ammonium-fed rice leaves. The enzyme assays showed that the activities of the two anti-oxidative enzymes, catalase and peroxidase, and the content of a non-enzymic antioxidant, glutathione, were significantly higher in the ammonium-fed rice leaves than those in the nitrate-fed ones, indicating that the ammonium nutrition might be beneficial for rice plants to improve the stress resistance during growth and development.

Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display

mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers （20 sets in total） were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ESTs were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ESTs were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ESTs were prepared for selecting genes related to adipose cells.

Analysis of Genetic Diversity and Relationships of Seven Chinese Indigenous Pig Breeds and Three Exotic Pig Breeds Using the DNA Differential Display Technique

The genetic diversity and relationships of seven Chinese indigenous pig breeds （Meishan, Erhualian, Hezuo, Bamei, Qingping, Tongcheng, and Huainan） and three exotic pig breeds （Large White, Landrace, and Duroc） were analyzed using the DNA differential display technique by means of eight primer combinations. A total of 123 reproducible bands were used to calculate mean Nei＇s gene diversity, and mean Shannon＇s information index for each pig population. Based on these the Nei＇s standard genetic identity and distance were estimated, which was used to construct a dendrogram tree for the 10 pig breeds. The experimental results obtained and the method used in this study for evaluating the genetic diversity and relationships of pigs were also discussed.

mRNA differential display on gene expression in settlement metamorphosis process of Ruditapes philippinarum larvae

The mRNA differential display (DDRT-PCR) technique was adopted to find out the genes related tosettlement metamorphosis development process of Ruditapes philippinarum larvae.In this study,we haveobtained three hundred and forty-six amplification bands in total from pediveliger larvae,veliger larvae,eye spot larvae and post-larvae.Sixty-five out of three hundred and forty-six bands are distinctly differen-tial display from band pattern,which can be put into four groups,standing for different expression char-acters.Sixteen differential display bands were cloned,sequenced and analyzed and nine different se-quences are obtained in the study.Three sequences have higher similarity to the cDNAs deposited indatabase and three are very similar to the rDNA of other species,considered as the rDNA of Ruditapesphilippinarum.The rest three sequences are found to be novel sequences after analyzed.Their accessionnumbers are AY916799,AY916798,and AY916797 respectively.We thought the novel sequences arepossibly relevant to the early embryo development of Ruditapes philippinarum larvae and can provide somefundamental understandings that are helpful for the improvement of scallop seed raising industry.

Identification and expression of a novel human testis-specific gene by digital differential display

Background Evidence for the importance of genetic factors in male infertility is accumulating This study was designed to identify a novel testis specific gene related to spermatogenesis by a new strategy of digital differential display (DDD) Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene Multi tissue RT PCR was performed to analyse its tissue specific expression The full length cDNA of the new gene was obtained using the BLAST program Sequencing was performed and the result was analysed Semi quantitative RT PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene The sequence of the opening reading frame was integrated into the pQE 30 vector expressed in Escherichia coil strain M15(pREP4) With IPTG induction, the target protein was detected Results A full length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified SPATA12 was 2430 bp in length, located in chromosome 3p21 1 3p21 2 The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT PCR and sequencing The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20 417 8 and isoelectric point of 5 23 The sequence has no significant homology with any known protein in databases Semi quantitative RT PCR and Northern blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis The expression recombinant of SPATA12 was constructed and a high level of the histidine tagged fusion protein was obtained Conclusions DDD can be confirmed by SPATA12 as a novel computational biology based approach for identification of the testis specific expression genes SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein

Screening for glutamate-induced and dexamethasone-downregulated epilepsy-related genes in rats by mRNA differential display

Background It is known that excessive release of glutamate can induce excitotoxicity in neurons and lead to seizure. Dexamethasone has anti-seizure function. The aim of this study was to investigate glutamatedexamethasone interaction in the pathogenesis of epilepsy, identify differentially expressed genes in the hippocampus of glutamate-induced epileptic rats by mRNA differential display, and observe the effects of dexamethasone on these genes expression. Methods Seizure models were established by injecting 5μl （250 μg/μl） monosodium glutamate （MSG） into the lateral cerebral ventricle in rats. Dexamethasone （5 mg/kg） was injected intraperitoneally at 30 minutes after MSG inducing convulsion. The rats＇ behavior and electroencephalogram （EEG） were then recorded for 1 hour. The effects of dexamethasone on gene expression were observed in MSG-induced epileptic rats at 1 hour and 6 hours after the onset of seizure by mRNA differential display. The differentially expressed genes were confirmed by Dot blot. Results EEG and behaviors showed that MSG did induce seizure, and dexamethasone could clearly alleviate the symptom, mRNA differential display showed that MSG increased the expression of some genes in epileptic rats and dexamethasone could downregulate their expression. From more than 10 differentially expressed eDNA fragments, we identified a 226 bp eDNA fragment that was expressed higher in the hippocampus of epileptic rats than that in the control group. Its expression was reduced after the administration of dexamethasone. Sequence analysis and protein alignment showed that the predicted amino acid sequence of this cDNA fragment kept 43% identity to agmatinase, a member of the ureohydrolase superfamily. Conclusions The results of the current study suggest that the product of the 226 bp eDNA has a function similar to agmatinase. Dexamethasone might relax alleviate seizure by inhibiting expression of the gene.

Differential Display of Cotton cDNAs Expressed by Salicylic Acid Induction

Salicylic acid (SA) is very important in systemic acquired resistance and hypersensitive response in plant defense, and yet its role is not fully understood. This study seeks to clarify the mechanism of SA induced resistance in cotton. Total RNA was extracted from low gossypol cultivated cotton seedlings treated with exogenous SA and subjected to fluorescent differential display PCR (FDD PCR). Seven cDNA fragments were selected from the total ten differential bands. Comparison with Genbank database shows that all seven cDNA sequences are newly discovered in cotton. However, they share high amino acid identity to some registered cDNAs. Among them, three of the cDNAs could be predicted to encode basic chitinase, penicillin binding 6 b precursor and ATP dependent DNA helicase RecG, while the functions of the other four cDNAs are undetermined. Dot blot analysis demonstrates that the expression of five cDNAs in cotton seedlings is induced by SA, while SA induction has a negative effect on the transcript accumulation of the other two cDNAs (E13 and E14). Since SA was previously shown to enhance the resistance to cotton wilt disease, the finding of a basic chitinase gene in cotton expressed by SA induction will provide a new insight into induced disease resistance in cotton.

Preliminary studies on tin miners’ lung cancer tissue related genes by differential display mRNA

Objective To study the genes related to tin miners' lung cancer tissue Method Differential display mRNA Results Thirty cDNA fragments which differentially expressed in lung cancer tissues and the same patient's normal lung tissues were discovered Among these, 16 expressed in lung cancer tissues, not in normal lung tissues; fourteen expressed on the contrary Six cDNA fragment sequence was determined Five sequences CG2, CG7, CG8, CA5 and CC6 had less than 75% homology with known sequences in GenBank BLAST, so they were believed to be new sequences which we have recorded in Genbank Only one fragment coded CG3 had homology up to 95% with human ribosome protein L27a gene Conclusions mRNA differential display provides a unique and powerful experimental system to study differential gene expression in tin miners' lung cancer tissues and the same patient's normal lung tissues Using the system, differential expression of 30 cDNA fragments was observed Six of them may be used to study the molecular mechanism of miners' radon associated lung cancer

Stage-specific expression genes of the Spirometra erinaceieuropaei plerocercoid screened by mRNA differential display technique

Background The stage-specific expression of genes is o ne of the most characteristics of parasites. It has been found that a lot of gen es of Spriometra erinaceieuropaei are specifically expressed in pleroceroid in large amount, but not expressed when the plerocercoid development into adult worm. The study is to screen other stage-specific ecpression genes of plerocerc oid of Spirtmetra erinceieuropaei.Methods RNA was separately extracted by acid guanidinium thiocyanate-phenol-chloroform from plerocercoids and adult worms of Spirometra erinaceieuropaei, DN A contaminated in the RNA was digested by RNase-free DNase. After the RNA was reverse transcripted to cDNA using T 12 MA, T 12 MC, T 12 MG and T 12 MT anchor-primers, PCR was done using the same T 12 MN and one random primer with α 35 S-dATP in the system. The PCR products were fractionated on an 8% denatured polyacrylamide gel. Differential bands of the plerocercoid found in the gel were cut out, amplified by PCR and sequenced. Northern hybridization was used to identify the stage-specific expression genes.Results Eleven differential bands were selected from the gel and classified into 3 kinds of gene fragments by hybridization, after they were amplified by PCR. Fragments 1 (238 bp) and 2 (383 bp), were confirmed by Northern hybridization, as being expressed in the plerocercoid. However, fragment 3 (433 bp), was expressed in both the plerocercoid and the adult worm. Data from the 3 gene fragments underwent homological analysis in GenBank. The sequence which was homologous with fragments 1 and 2 was not found, but fragment 3 had high homology with many kinds of 28S rRNA. Conclusions The gene expressions of plerocercoids are different from adult worms because they live in different hosts. Two types of different gene fragments from the plerocercoid were found by mRNA differential display technique.

Differential Gene Expression Between Wheat Hybrids and Their Parental Inbreds in Primary Roots

To provide an insight into the molecular basis of heterosis, differential display of mRNA was used to analyze the difference of gene expression between wheat (Triticum aestivum L.) heterotic hybrid A, nonheterotic hybrid B and their parental inbreds in the primary roots. By using 5′ end random primers in combination with three one-base-anchored primers, it was found that 22.5% and 22.9% of 877 total displayed cDNAs were differentially expressed between hybrid A, B and their parents, respectively. Both quantitative and qualitative differences in gene expression between hybrids and their parental inbreds were obvious, indicating that the patterns of gene expression in hybrids alter significantly as compared to their corresponding parents. On the other hand, by using MADS-box gene specific 5′ end primer for DDRT-PCR, we found that nearly all of the displayed cDNA fragments were polymorphic between hybrids and their parents, and major difference occurred in qualitative level, in which hybrid specific-expressed and silenced genes are the major two patterns, suggesting that MADS-box gene may be important for manifestation of differential gene expression and wheat heterosis. In comparison with our previous results by using seedling leaves, it is indicated that differential gene expression between hybrids and parents is dependent on the tissues tested, and more differentially expressed genes were observed in the primary roots than in the seedling leaves. Therefore, it is concluded that the expressions of both randomly displayed cDNAs and transcription factor genes, such as MADS-box, alter significantly between hybrids and their parents, which might be responsible for the observed heterosis.

Identification of differentially expressed genes in dorsal root ganglion in early diabetic rats

Objective To screen and identify differentially expressed genes in the dorsal root ganglion （DRG） in early experimental diabetic rats. Methods Diabetic model rats were induced by single intraperitoneal injection of streptozotocin （STZ）. At the second week after STZ injection, the sensory nerve conduction velocities （SNCV） of sciatic nerve were measured as an indicator of neuropathy. The technique of silver-staining mRNA differential display polymerase chain reaction （DD-PCR） was used to detect the levels of differentially expressed genes in rat DRG. The cDNA fragments that displayed differentially were identified by reverse-hybridization, cloned and sequenced subsequently, and then confirmed by Northern blot. Results The SNCV in the diabetic model group [n = 9, （45.25±10.38） m/s] reduced obviously compared with the control group [n = 8, （60.10± 11.92） m/s] （P 〈 0.05）. Seven distinct cDNA clones, one was up-regulated gene and the others were downregulated ones, were isolated by silver-staining mRNA differential display method and confirmed by Northern blot. According to the results of sequence alignment with GenBank data, majority of the clones had no significant sequence similarity to previously reported genes except only one that showed high homology to 6-pyruvoyl-tetrahydropterin synthase mRNA （accession No., BC059140）, which had not been reported to relate to diabetic neuropathy. Conclusion These differentially expressed genes in the diabetic DRG may contribute to the pathogenesis of diabetic peripheral neuropathy.

Relationship Between Differential Gene Expression and Heterosis During Ear Development in Maize (Zea mays L.)

Maize （Zea raays L.） is one of the most important crops because of the remarkable properties of its hybrid, which is responsible for the high commercial value of hybrid maize. The genetic basis of heterosis （hybrid vigor） is not well understood. A differential display technique was performed to identify genes with differential expression across twelve maize inbred lines and thirty-three hybrids during ear development. An incomplete diallel design was used to investigate the relationship between the global framework of differential gene expression and heterosis. It was found that the genes belonging to MONO pattern （i.e., genes expressed in both parental lines and in hybrid） was the highest in percentage among the total five patterns and illustrated that the properties of differentially expressed genes are not entirely responsible for heterosis. Furthermore,a larger number of differentially expressed genes in hybrid, which serves as a major reservoir for generating novel phenotypes that exhibit heterosis of certain agronomic traits during early development and differentiation of maize ear. Moreover, there were some silent genesin hybrids that are responsible for the arrest or abortion of spikelets and for the increase in kernels weight.

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer

Objective： To screen and analyze key express sequence tags （ESTs） which were differentially displayed in every period of SD rats＇ primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods： Using diethylnitrosamine （DENA） as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique （DDRT-PCR）. After the fragments were sequenced, bioinformatics were .used to analyze the results. Results： Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments （EST-1, EST-3 and EST-5） had extremely low identity to any genes registered in GENBANK databases. Conclusions： BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-I, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Differential Gene Expression Between Hybrids and Their Parents During the Four Crucial Stages of Cotton Growth and Development

The study aims to clarify the differential gene expression between cotton hybrids and their parents in order to better understand the molecular basis of cotton heterosis. The research focused on cotton heterotic and lower heterotic hybrids and their parents during the four crucial stages, which were analyzed using a differential display technique. The results indicated that there were both quantitative and qualitative differences in gene expression amongst them. The quantitative differences include over- and under-expression of parental genes and the dominant expression of highly-expressed parental genes in hybrids. In contrast, the qualitative differences are the following： （i） Bands were observed in both parents but not in the F1 hybrid （BPnF1）; （ii） bands occurred in either of the parents but not in the F1 hybrid （UPnF1）; （iii） bands presented only in the F1 hybrid but not in either of the parents （UF1nP）; and （iv） bands were detected in either of the parents and the F1 hybrid （UPF1）. Overall, the major differences of gene expression occurred in the qualitative level and four related differential patterns were observed. Furthermore, the amount of differential patterns during the flowering stage was relatively higher than those of other stages. At this juncture, both the amount of hybrid-specific expression patterns at flowering stage and the silenced expression patterns at boll-forming stage in highly heterotic hybrids were found higher than those in the lower heterotic ones. It was concluded that significant differences of gene expression in leaves were present between cotton hybrid and its parents during the whole growing stages. Hence, these differences might be responsible for the observed cotton heterosis.

Genes Differentially Expressed in Human Lung Fibroblast Cells Transformed by Glycidyl Methacrylate

Objective To define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls. Methods The mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis. Results Eighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor b inducible gene (Betaig-h3), a-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells. Conclusion Analysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Isolation, Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan, Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3000 reproducible bands were examined. One novel expressed sequence tag (EST4, GenBank accession number: AY553914) that was differentially expressed in Meishan, Meishan× Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes. Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.LIU Yong-gang, Ph D candidate