Here, we investigated antioxidant defense mechanism in the spermatheca of A. mellifera queens via RNA-seq analysis of spermathecae in both mated and virgin queens. We identified the genes encoding antioxidant proteins, which were differentially expressed in the spermatheca of mated queens. The concentrations of antioxidant proteins, such as superoxide dismutase 1 (SOD1), catalase, glutathione peroxidase (GTPX), and transferrin (Tf) together with the levels of ROS, H2O2, and iron were higher in the spermathecal fluid of mated queens as opposed to those in the spermathecal fluid of virgin queens; this indicated that increase in antioxidant protein concentration is an antioxidant defense mechanism occurring in the spermathecal fluid of mated queens against ROS; this mechanism involves conversion of ROS using antioxidant enzymes and Tf-mediated inhibition of the Fenton reaction occurring between Fe2+ and H2O2. Our data indicate that an increased expression of antioxidant proteins could facilitate prolonged storage and survival of sperms in the spermatheca of mated queens, suggesting the role of antioxidant proteins in antioxidative defense against ROS.

Honeybee (Apis mellifera) egg-yolk protein vitellogenin (Vg) plays roles in immunity, antioxidation, and life span beyond reproduction, but it also acts as an allergen Api m 12 in venom. Here we established antimicrobial and antioxidant roles of honeybee Vg in the body and venom. Using the cDNA encoding Vg identified from Asiatic honeybee (A. cerana) workers, recombinant A. cerana Vg (AcVg) protein of approximately 180 kDa was produced in baculovirus-infected insect cells. In A. cerana worker bees, AcVg was expressed in the fat body and venom gland and was present in the secreted venom. AcVg induced structural damage in microbial cell walls via binding to microbial surfaces and exhibited antimicrobial activity against bacteria and fungi. AcVg protected mammalian and insect cells against oxidative damage through direct shielding of cell membranes. Interestingly, AcVg exhibited DNA protection activity against reactive oxygen species (ROS). Furthermore, the transcript level of AcVg was upregulated in the fat body, but not in the venom gland, of worker bees with antimicrobial peptides and antioxidant enzymes in response to microbial infection and oxidative stress. Our data indicate that AcVg is involved in innate immunity upon infection and in a defense system against ROS, supporting a crucial role of honeybee Vg as an antimicrobial and antioxidant agent in the body and venom.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

운간초의 분화 품질을 향상시키기 위하여 ‘Kumoma’ 와 ‘Kumoma-Gusa’ 두 품종의 생장과 개화에 미치는 영향을 구명하고자 식물생장억제제 (PGRs) 4종을 처리 하였다. 식물생장억제제 종류와 처리농도는 paclobutrazol (10, 20, 40, 80 mg·L-1), flurprimidol (5, 10, 20, 40mg·L-1), daminozide (500, 1000, 2000, 4000mg·L-1), chlormequat (50, 100, 200, 400 mg·L-1)이고 엽면살포 와 토양관주 방법으로 처리하였다. Paclobutrazol 40 mg·L-1 처리는 운간초의 두 품종에서 초장과 화경 장을 줄이는데 효과적이었다. ‘Kumoma’ 품종에서, paclobutrazol 40 mg·L-1 엽면살포와 토양관주 처리방 법은 초장을 각각 12.6, 12.5 cm로, 화경장은 3.4, 3.3 cm로 줄일 수 있었다. ‘Kumoma-Gusa‘ 품종에서 는 paclobutrazol 토양관주는 처리 농도가 증가함에 따라 초장은 13.2-10.7 cm, 화경장은 3.9-2.0 cm까지 줄일 수 있었지만 꽃의 수는 대조구와 비교했을 때 현 저하게 감소하여서 20개까지 감소하였다. Flurprimidol 20mg·L-1 스프레이와 토양관주 처리는 두 품종 모두 초장과 화경장을 줄일 수 있었다. ‘Kumoma’ 품종에 서 flurprimidol 10 mg·L-1 스프레이 또는 토양관주 처리는 개화 품질을 향상시킬 수 있었고 대조구와 비 교해 꽃의 수는 44.7개 이상, 출하 일수는 5일 정도 빨리 출하 할 수 있었다. ‘Kumoma-Gusa‘ 품종에서 PGRs처리에 의한 초장 과 화경장 억제효과가 가장 큰 것은 paclobutrazol 80mg·L-1과 flurprimidol 40mg·L-1 이었고 paclobutrazol 과 flurprimidol 스프레이와 토양관주 처리에서 각각, 초장은 10.7, 9.9 cm, 화경장은 2.0, 1.5 cm 로 생장 억제효과가 가장 큰 것을 알 수 있었다. 그러나 flurprimidol 40 mg·L-1 토양관주 처리에서 출하일수는 농도에 따라 3-13일 정도 대조구보다 늦어졌고 꽃의 수는 15.6개로 가장 적은 것을 관찰 할 수 있었다. 두 품종 모두에서 daminozide 처리는 초장과 화경장 이 줄어들지 않았지만 ‘Kumoma’ 품종에서는 대조구 와 비교했을 때 꽃의 수가 증가하였다. Chlormequat 처리는 paclobutrazol 과 flurprimidol 처리와 비교했 을 때 화경장 감소효과는 작았고 처리 식물의 잎은 다른 처리에 비해 연한 녹색을 보여주었다. 두 품종 모두에서 chlormequat 과 daminozide 처리는 생육과 개화 품질에 효과적으로 영향을 주지는 않았지만 꽃의 수는 모든 처리농도에서 41개 이상을 보여주었고, ‘Kumoma-Gusa’ 품종에 chlormequat 토양관주 처리는 최대 꽃의 수 63개를 볼 수 있었다. 이번 결과들은 paclobutrazol 과 flurprimidol 처리가 운간초 분화의 품질 향상을 위한 화경장의 생육을 조 절하는 생장억제제로서 사용 될 수 있음을 보여 주었 고, ‘Kumoma’ 품종에서는 paclobutrazol 40 mg·L-1 스프레이는 생육특성을, flurprimidol 10 mg·L-1 토양관 주 방법은 개화관련 특성의 품질을 향상시키는 생장억 제제의 농도로 추천 될 수 있고, ‘Kumoma-Gusa’ 품종 에서는 flurprimidol 20mg·L-1 스프레이와 flurprimidol 5mg·L-1 토양관주 방법이 운간초 분화의 생육특성과 개 화품질을 향상시키는 생장억제제의 농도로 추천 될 수 있다.

Rye has important genes for biotic and abiotic stress resistance. Introduction of these genes to wheat by breeding wheat-rye translocation have been intensively used in wheat breeding program. Rye chromatin 1RS and/or 2RL show superior performance in unfavorable environments. In order to develop high yielding wheat, we applied various molecular breeding strategies. To develop EST-derived 1RS specific markers, we used comparative genomics with public sequence databases of Poaceae family. Putative rye chromatin specific sequences were used to design 1RS specific markers. To identify genes related to water deficiency, cDNA AFLP analysis was used in PEG treated seedlings of 1RS RILs. For functional analysis of identified genes and markers, we used Brachypodium distachyon, as a new model plant of temperate grasses. B. distachyon were recently applied for transformation and we constructed Agrobacterium-mediated transformation system. Integration of those strategies and conventional breeding method would enhance the usefulness of rye chromatins for wheat improvement.