木豆杂种优势利用研究进展

自1974年发现木豆雄性不育株以来,国际上不断对木豆核雄性不育(GMS)和核质互作雄性不育(CGMS)基因及其种质资源进行研究和挖掘,最终实现了木豆杂种优势的成功利用。本文系统总结了过去30多年来,国际上木豆杂种优势机理、表现及授粉方式等基础研究,GMS、CGMS不育系及其配套制种体系研究,杂种优势强度研究,以及木豆杂交种生产技术研究进展。提出了木豆杂种优势利用的具体研究方向和展望。     

Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae)

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations.     

Identification of Polymorphisms in the Osteopontin Gene and Their Associations with Certain Semen Production Traits of Water Buffaloes in the Brazilian Amazon

The osteopontin gene may influence the fertility of water buffaloes because it is a protein present in sperm. The aim of this work was to identify polymorphisms in this gene and associate them with fertility parameters of animals kept under extensive grazing. A total of 306 male buffaloes older than 18 months, from two farms, one in the state of Amapá and the other in the state of Pará, Brazil were used in the study. Seven SNPs were identified in the regions studied. The polymorphisms were in gene positions 1478, 1513 and 1611 in the region 5′upstrem and positions 6690, 6737, 6925 and 6952 in the region amplified in intron 5. The SNPs were associated with the traits, namely scrotal circumference, scrotal volume, sperm motility, sperm concentration and sperm pathology. There were significant SNPs (p < 0.05) for all the traits. The SNP 6690 was significant for scrotal circumference, sperm concentration, sperm motility and sperm pathology and the SNP 6737 for scrotal volume. The genotype AA of SNP 6690 presented the highest averages for scrotal circumference, sperm concentration and motility and the lowest total number of sperm pathologies. For the scrotal volume trait, the animals with the largest volume were correlated with the presence of the genotype GG of SNP 6737. These results indicate a significance of the osteopontin gene as it seems to exert a substantial influence on the semen production traits of male buffaloes.     

Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties

A 2193-nucleotide cDNA encoding acetylcholinesterase (AChE) of the stable fly, Stomoxys calcitrans (L.) was sequenced and expressed in the baculovirus system. The open reading frame encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 96% and 94% identity to the AChEs of Haematobia irritans (L.) and Musca domestica (L.), respectively. Structural characteristics of M. domestica, H. irritans, and Drosophila melanogaster AChEs were conserved in the S. calcitrans AChE. The recombinant enzyme was inhibited by eserine, coroxon, and paraoxon and exhibited K_m values of 63.9 μM for acetylthiocholine and 96.7 μM for butyrylthiocholine, confirming its biochemical identity as an acetylcholinesterase (EC 3.1.1.7). These data will enable rapid identification and assay for mutations that reduce AChE sensitivity to organophosphate (OP) pesticides, potentially aiding resistance management efforts to prevent fixation of the mutations in pest populations.     

Antigenicity and immunogenicity of Hypoderma lineatum soluble proteins in the bovine host

Protein species found in soluble crude extracts of Hypoderma lineatum (common cattle grub) 1st-instar larvae (HL1) were separated by non-denaturing and denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed for antigenicity by Western blotting using serum from H. lineatum-infested and vaccinated cattle. All HL1 proteins resolved by non-denaturing PAGE were found to be antigenic in the infested bovine host. Treatment of the proteins with sodium dodecyl sulfate and 2-mercaptoethanol destroyed the ability of hypodermin B and the Peak 2 proteins from DEAE-ion exchange HPLC to be bound by antibody. The principal proteins, hypodermin A and hypodermin C (collagenase), appear to be the most immunogenic of the larval proteins. Although having similar amino acid composition, hypodermin A did not appear to share an antigenic epitope with the most prevalent protein, hypodermin C. These results may allow for the selection of proteins to be used in vaccine trials and studies of protective immunological mechanisms associated with acquired resistance to H. lineatum infestation in the bovine host.     

Colostral transfer of antibodies specific for Hypoderma lineatum proteins

Antibodies to Hypoderma lineatum were transferred to calves via colostrum. The antibodies transferred in the colostrum demonstrated specificity to all the H. lineatum first-instar proteins which were resolved by non-denaturing polyacrylamide gel electrophoresis and were capable of mediating a Type I hypersensitivity reaction. The kinetic decline of colostrum-acquired antibodies, the effect upon development of an autologous humoral response to H. lineatum and the host or parasite protective role of these antibodies are discussed.     

Identification of lactoferrin and glutamate receptor‐interacting protein 1 in bovine cervical mucus: A putative marker for oestrous detection

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous‐specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non‐oestrous and controlled internal drug release (CIDR)‐induced oestrous‐stage mucus proteins were purified and subjected to surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI‐TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor‐interacting protein 1 (GRIP1) showed a twofold increase during the CIDR‐induced oestrous stage compared to the levels in non‐oestrous stage in bovine cervical mucus. The RT‐PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non‐oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.