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.

Differential Gene and Protein Expression in Soybean at Early Stages of Incompatible Interaction with Phytophthora sojae

Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP b gene coding ATP synthase b subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-a-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, a-subunit and b-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.

Molecular cloning and identification of naturally occurring human antisense angiopoietin-1: Gna-1

One novel cDNA fragment was obtained from vascular endothelial cells by differential display reverse transcription PCR technique. By using this fragment as probe, we screened the human artery cDNA library and obtained one cDNA clone which is 2198 bp in length. After sequencing and homology researching, we found that the clone contained a region of 851 bp in length complementary to that of human angiopoietin-1 cDNA, encoding the partial fibrinogen-like domain and 3′ non-translational region. It was inferred that this clone was a naturally occurring antisense RNA of human angiopoietin-1, designated as Gna-1. Gna-1 does not encode protein. The tran-scription of Gna-1 in human umbilical vein endothelial cells and ECV304 cells was confirmed by RT-PCR method. Gna-1 may be involved in regulating the function of angiopoietin-1, and play a significant role in angiogenesis.