Lipopolysaccharide binding protein (LBP) is a key factor in the recognition of lipopolysaccharide (LPS) and the initiation of immune response, thus regulating the body’s resistance to pathogenic infection.  To investigate the tissue-specific expression characteristics of the LBP gene and its transcriptional regulation in pigs, we detected LBP expression in different tissues of 35-day-old Meishan weaned piglets, determined LBP core promoter region using bioinformatics prediction combined with dual luciferase activity assay, and finally detected methylation levels by pyrosequencing.  The results showed that LBP expression in the liver tissue was significantly higher (P<0.01) than that in other tissues, followed by the intestinal tissues.  The core promoter region of LBP was located at –500–(–206) bp (chr.17: g.46837534–g.46837828), containing three CpG sites (CpG1, CpG2 and CpG3).  Of the three CpG sites, CpG2 and CpG3 were variously methylated (P<0.01) in different tissues.  Moreover, LBP mRNA levels were negatively correlated (P<0.01) with methylation levels of the CpG2 and CpG3 sites in the YY1 transcription factor binding sequence.  It is speculated that the methylation of CpG2 and CpG3 sites might inhibit YY1 binding to the promoter sequences, thereby regulating the tissue-specific expression of LBP.  This study demonstrated the distinct patterns of LBP expression and promoter methylation in the tissues of Meishan pigs and indicated the potential roles of DNA methylation in regulating LBP expression, which may contribute to further investigations on pig LBP gene expression and function. 

 

The bactericidal/permeability increasing protein (BPI) has an important function of nonspecific killing of Gram-negative bacteria.  In this study, qPCR was used to detect the expression of the BPI gene in twelve tissues of Meishan piglets from birth to weaning.  BPI gene overexpression, bacterial adhesions count and indirect immunofluorescence were applied to analyze the relationship between BPI gene expression and the infectivity of Escherichia coli and Salmonella.  The results showed that the BPI gene was expressed highly in duodenum, jejunum and ileum (fold changes of relative expression levels were more than 10 000, 500 and 200, respectively).  The expression of the BPI gene at 35 days of age was significantly higher (P<0.01) than that at all other days.  Transcription of the BPI gene was up-regulated 2 401-fold in porcine intestinal epithelial (IPEC-J2) cells that were transfected with the BPI gene overexpression lentivirus (IPEC-J2-BPI), and significantly higher (P<0.01) than that in negative control cells (IPEC-J2-NC).  Protein expression levels in IPEC-J2-BPI cells were also increased.  When IPEC-J2 cells were incubated with E. coli and Salmonella, respectively, for 2, 4, 6, 8, 10 and 12 h, the number of bacterial adhesions in IPEC-J2-BPI cells was significantly less (P<0.05) than that in IPEC-J2-NC cells.  The results of indirect immunofluorescence analysis showed that the number of bacterial adhesions in IPEC-J2-BPI cells was significantly less (P<0.01) than that in IPEC-J2-NC cells.  These results demonstrated that the BPI gene might play an important role in regulating weaning stress especially intestinal-mediated immune response.  Overexpression of the BPI gene at the cellular level could significantly enhance the anti-bactericidal ability against Gram-negative bacteria such as E. coli and Salmonella.  This has important biological significance in piglet resistance to bacterial diarrhea. 

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-mediated pathway of shikimic acid.  The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop.  Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS.  In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32.  Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node.  PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels.  In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha^–1 of glyphosate.  All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.