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.

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.

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.

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.

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

Isolation, Identification of Differentially Expressed Sequence Tags in the Backfat Tissue from Meishan, Large White and MeishanLarge White Cross Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performedto investigate the differences of gene expression in the backfat tissue from Meishan, Large White and MeishanLargeWhite cross pigs. Nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers were used to perform thedifferential display PCR and nearly 3 000 reproducible bands were examined. Fifteen expressed sequence tags that weredifferentially expressed were isolated and then identified through semi-quantitative RT-PCR. BLAST analysis revealedthat the fifteen expressed sequence tags (ESTs) were not homologous to any of the known porcine genes or ESTs. Thesenovel ESTs were then submitted to GenBank.

ISOLATION AND IDENTIFICATION OF cDNA FRAGMENTS AND FULL-LENGTH cDNA DIFFERENTIALLY EXPRESSED IN HUMAN GLIOBLASTOMA CELL LINE BT-325 VERSUS ALL-TRANS RETINOIC ACID INDUCTION

Objective.To investigate the differentiation process of the human glioblastoma cells. Methods.Differential display reverse transcribed-PCR(DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325.Routine method of cDNA library screening was performed to clone full-length cDNA. Results.Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered,amplified and cloned.Of them,46 ESTs were sequenced and delivered into the GenBank.The homology comparison using BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play important roles in the cell differentiation progress.A dot-blot hybridization was conducted to certify the differentiation expression.The result showed that 27 EST clones are expressed at different level in control and all-trans retinoic acid treated BT-325 cells.A full-length cDNA was cloned using the EST-HGBB098. Conclusion.DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

Effect of Epinephrine on the Settlement and Metamorphosis of Manila Clam Larvae

Chemical inducement and DDRT-PCR （differential display reverse transcription PCR） are adopted to investigate the effect of epinephrine （EPI） on the settlement and metamorphosis of Manila clam larvae. Chemical inducement shows that EPI has an effect to some extent on the metamorphosis of Manila clam larvae at all concentrations and in all treatments designed. The most significant result of inducement is obtained at the concentration of 10^-6 tool L^-1 and for 4 h. DDRT-PCR using six primer pairs shows that the gene expression pattern is quite different between EPI treatment and the control. Three hundred and forty-three amplification bands are obtained in total, among which, 67 （19.53%） are differentially appeared. Therefore, EPI has an effect on the gene expression of the eye spot larval Manila clam. It can be hypothesized that EPI is a settlement and metamorphosis inducer for Manila clam. EPI may lead to larvae settlement and metamorphosis by binding to the receptors on the membrane and then changing the gene expression of larvae cells.