Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Comparative analyses of the venom components in the salivary gland transcriptomes and saliva proteomes of some heteropteran insects

Salivary gland-specific transcriptomes of nine heteropteran insects with distinct feeding strategies (predaceous, hematophagous, and phytophagous) were analyzed and annotated to compare and identify the venom components as well as their expression profiles. The transcriptional abundance of venom genes was verified via quantitative real-time PCR. Hierarchical clustering of 30 representative differentially expressed venom genes from the nine heteropteran species revealed unique groups of salivary gland-specific genes depending on their feeding strategy. The commonly transcribed genes included a paralytic neurotoxin (arginine kinase), digestive enzymes (cathepsin and serine protease), an anti-inflammatory protein (cystatin), hexamerin, and an odorant binding protein. Both predaceous and hematophagous (bed bug) heteropteran species showed relatively higher transcription levels of genes encoding proteins involved in proteolysis and cytolysis, whereas phytophagous heteropterans exhibited little or no expression of these genes, but had a high expression of vitellogenin, a multifunctional allergen. Saliva proteomes from four representative species were also analyzed. All venom proteins identified via saliva proteome analysis were annotated using salivary gland transcriptome data. The proteomic expression profiles of venom proteins were in good agreement with the salivary gland-specific transcriptomic profiles. Our results indicate that profiling of the salivary gland transcriptome provides important information on the composition and evolutionary features of venoms depending on their feeding strategy.