Cloning and characteristics of the antibacterial peptide gene abaecin in the bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Among the antimicrobial peptides, abaecin is rich in proline content and plays a vital role in insect innate immune defense. Here, the full-length gene of abaecin from the bumblebee Bombus lantschouensis was cloned, and its expression profiles for different tissues, developmental stages and reproductive statuses were analyzed by RT-qPCR. Meanwhile, the responses of abaecin to a bacterium (Escherichia coli) and a fungus (Beauveria bassiana) were tested. The full length of abaecin cDNA was 470 bp, and the open reading frame (ORF) was 258 bp, encoding a polypeptide of 85 amino acids. The abaecin gene consists of three exons and two introns. Phylogenetic analysis showed that Bombus ignitus was the closest species to B. lantschouensis base on putative Abaecin protein sequence. Expression analysis showed that abaecin was expressed broadly in different tissues, with the highest expression in fat bodies and extremely low expression in antennae. Regarding developmental stage, low expression of abacein was detected in eggs and larvae, and high expression was detected in pupal stages. The highest expression was observed at the Pw pupal stage (pupae with an unpigmented body cuticle and white eyes), and the expression then decreased from the Pp (pupae with pink eyes) to the Pdd (dark-eye pupae with a dark-pigmented cuticle) stages. In addition, the expression of abaecin was higher in egg-laying than in non-egg-laying female bumblebees. Both E. coli and B. bassiana infections induced the expression of abaecin. Our results indicated that the abaecin gene plays important roles in the development, reproduction and immune responses of bumblebees. During the artificial rearing of bumblebees, a good environment should be created to avoid infection with bacteria or fungi.     

Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris

Three major components of bumblebee venom are bombolitin, phospholipase A₂, and a serine protease, with bombolitin being the most abundant. Here, we describe the molecular cloning of bombolitin isolated from the venom of a bumblebee, Bombus terrestris, and demonstrate its antibacterial activity. The B. terrestris bombolitin gene consists of 2 exons encoding 56 amino acid residues. Comparative analysis shows that mature B. terrestris bombolitin consists of 18 amino acid residues, which are identical to those of B. ignitus bombolitin. B. terrestris bombolitin displayed antibacterial activity against both the Gram-negative bacterium Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus, indicating that B. terrestris bombolitin may be a potential antimicrobial agent.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

红光熊蜂卵黄原蛋白基因的cDNA全长序列克隆和表达分析

红光熊蜂Bombus ignitus Smith是许多经济作物和野生植物的重要授粉昆虫之一。卵黄原蛋白基因(vitellogenin,Vg)在昆虫的生殖调控中和行为方面起到重要的作用,本试验对Vg基因全长cDNA的克隆和测序及在蜂王、工蜂和雄性蜂三型蜂中的表达分析得出:Vg基因的全长cDNA为5 481 bp,GenBank中的登录号为FJ913883,有一个完整的开放阅读框(ORF),编码1 772个氨基酸,N-末端的前16个氨基酸为一个信号肽。接近C-末端区域存在保守的GL/ICG基元,其后含有9个半胱氨酸,而且DGXR位于GL/ICG基元上游18个氨基酸残基处。其氨基酸序列与韩国的熊蜂B.ignitus和B.hypocrita相似性高达95%,与西方蜜蜂Apis mellifera的相似性达到51%。Vg的mRNA首先在蜂王蛹期的白眼蛹(Pw)时期出现,其表达量在蜂王整个蛹期发育过程中呈上升趋势,且在黑眼蛹(Pbd)时期达到最高,在成年蜂的脂肪体中的表达量仍在升高,甚至更高。Vg也在工蜂蛹期的白眼蛹(Pw)时期被检测到,然后在整个蛹期发育过程中呈现上升趋势,在刚羽化出房时达到高峰,Vg的mRNA水平随着成年蜂日龄的增加而增加,到15日龄时达到最高,然后呈现下降趋势。对于雄性蜂,Vg的mRNA虽然卵黄原蛋白基因的mRNA水平几乎在整个蛹期发育阶段都表达,但是表达水平非常低,只有在刚羽化出房时期表达水平较高。     

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking. The full-length DNA of the scallop H2A was 696bp long, including a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 228bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated.     

梭梭糖基转移酶基因克隆及序列分析

以梭梭叶片为材料,根据其他植物糖基转移酶基因的保守序列设计引物,采用同源基因克隆及RACE-PCR方法克隆到2个同源的序列,分别命名为 HaUGT1和 HaUGT2。基因测序结果显示, HaUGT1基因编码区长1 485 bp,编码495个氨基酸; HaUGT2基因编码区长1 185 bp,编码394个氨基酸。HaUGT1和HaUGT2蛋白具有保守的PSPG 基序、UDP-葡萄糖基转移酶结构域,与其他植物中的糖基转移酶蛋白同源性较高。     

Diploid male production in a rare and locally distributed bumblebee, Bombus florilegus (Hymenoptera, Apidae)

The European bumblebee B. terrestris was recently introduced in Japan for agricultural purposes and has now become naturalized. The naturalization of this exotic species may have great detrimental effects on closely related native Japanese bumblebees. The Japanese bumblebee Bombus florilegus is a rare and locally distributed species found in the Nemuro Peninsula of Hokkaido, Japan. In order to assess its population genetics, we estimated the genetic structure of B. florilegus in 16 breeding colonies (queen, workers, and males) and 20 foraging queens by analyzing microsatellite DNA markers. Of the 36 queens analyzed by genotyping and dissection, 32 had been inseminated by a male. The remaining 4 had not been inseminated at all. Of the 4 nonmated queens, one was triploid. Diploid males were found in 4 breeding colonies. Based on the microsatellite data, it appears that B. florilegus has low reproductive success. Since matched mating and nonmating within local populations are high, the extinction risk is correspondingly higher. Our results suggest that conservation of the Japanese B. florilegus is required in order to protect it from both habitat destruction and the naturalization of alien species. Received 19 July 2007; revised 13 October 2007; accepted 15 October 2007.     

Spatial and temporal pattern of introduced Bombus terrestris abundance in Hokkaido, Japan, and its potential impact on native bumblebees

A commercial colony of Bombus terrestris (L.) was introduced to Japan in 1992 for crop pollination in greenhouses. Since then wild colonies have developed and spread in some regions. In the present study, we measured the spatial distribution and temporal change in abundance of B. terrestris in the Chitose River Basin, Hokkaido, Japan to elucidate the relation of greenhouses to the bees distribution and to evaluate its potential effects on native bumblebees. Bumblebees were collected with window traps in windbreak forests roughly 1, 2, 4, and 6 km NNW and SSE of a large greenhouse. The peak catch of B. terrestris queens occurred in early June, suggesting that they had successfully hibernated in the field. The distributions of B. terrestris and the native B. ardens were mutually exclusive, while the native B. hypocrita appeared at all sites. Catches of B. terrestris were restricted to within 4 km of the nearest greenhouse, suggesting that the invasion was still in the initial phase in this area. The reduction in abundance of the native bumblebees in the sites of high B. terrestris abundance suggests the presence of interspecies competition between B. terrestris and the native bumblebees during the early part of the colony activity, although such reduction in B. ardens can be explained by habitat suitability.     

Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis

Summary DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33. The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance. A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made. The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size. Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations. The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments.     

抗菌肽CM4基因克隆及其在毕赤酵母中的表达鉴定

为了在毕赤酵母中获得抗菌肽CM4表达,将抗菌肽CM4基因克隆入表达质粒pPIC9,SacⅠ线性化的重组表达质粒pPIC9-CM4电击转化P.pastorisGS115(his-),采用MM、MD板和PCR法筛选Mut 表型,甲醇诱导表达;抗菌活性检测、Tricine-SDS-PAGE及酸性活性电泳均表明抗菌肽CM4在毕赤酵母中成功地分泌表达;重组抗菌肽CM4对黑曲霉(Aspergillus niger)、绿色木霉(Trichoderma viride)都有很强的抑菌活性。     

火红熊蜂微卫星标记的筛选及种特异性分析

火红熊蜂Bombus pyrosoma是中国特有的熊蜂资源, 在华北地区分布十分丰富, 是众多野生植物和农作物的重要传粉者。为了探究火红熊蜂的遗传结构及其进化关系, 首次开展了该种熊蜂卫星标记的筛选及其种特异性研究。本研究采用生物素-磁珠吸附分离法从火红熊蜂基因组中筛选微卫星标记, 并选取黑熊蜂亚属Melanobombus其他7种熊蜂进行了这些标记的种特异性验证。结果表明： 不同探针与磁珠杂交产生的微卫星标记阳性克隆率存在很大差异, TC探针的微卫星阳性克隆率最高为82%; TG探针次之, 为28%; AT和GC探针分离未获得微卫星标记克隆。根据获得的阳性克隆的序列设计引物进行筛选验证, 共获得31对熊蜂微卫星序列特异性引物。微卫星标记分析发现, 完美型(perfect)18个, 占58.1%; 非完美型(imperfect)10个, 占32.3%; 混合完美型(compound perfect)1个, 占3.2%; 混合非完美型(compound imperfect)2个, 占6.4%。不同探针的微卫星核心序列重复数不同, TC探针和TG探针的微卫星核心序列最高重复数分别为28和15个, 最低重复数分别为7和11个。利用所获得的31对微卫星引物对黑熊蜂亚属其他7种熊蜂进行检测, 有26对引物可以扩增所测试的7种熊蜂, 其他5对引物只能扩增部分熊蜂种类, 其中BPM5是火红熊蜂种特异性引物。本研究从火红熊蜂基因组中筛选的微卫星标记, 不仅可用于火红熊蜂的遗传结构、 分子进化和资源保护等方面的研究, 而且可进一步用于黑熊蜂亚属其他种群的遗传特性分析。     

Exon shuffling in anther-specific genes from sunflower

We describe here the nucleotide sequence of an anther-specific gene (sf18) from sunflower, encoding a proline- and glycine-rich polypeptide with a hydrophobic amino-terminus (signal peptide). The gene is split by a 211 by intron and is partially related to another anther-specific gene (sf2) from sunflower with which it shares important sequence stretches in the 5 coding and upstream regions. We propose that the two genes have originated via exon shuffling, during which a copy of a DNA segment including the promoter region as well as a signal peptide coding sequence has been transferred into the upstream region of two different potential coding sequences, generating two novel genes which display the same specificity of expression and which both encode an extracellular protein. While the 5 region of the intron is highly conserved as part of the transferred region and may play a role in the selection of the 5 splice site, a common octanucleotide at the 3 end of the intron of the two genes might be involved in 3 splice site selection.     

Cloning and analysis of the inulinase gene from Kluyveromyces cicerisporus CBS4857

A highly expressed inulinase gene, KcINU1 was cloned and sequenced from Kluyveromyces cicerisporus CBS4857 a strain which secrets high levels of inulinase into the growth medium. The result of DNA sequencing showed that KcINU1 contained a 1665 bp ORF, coding for a 555 amino acid protein, in which a 23 amino acid signal peptide was included. The sequence has the GenBank Accession no. AF 178979. The analysis of conserved domain in the ORF indicated there was a consensus sequence about 470 amino acids long. The 0.7 Kb promoter and 0.9 Kb terminator were also cloned and sequenced.     

Male labial gland secretions and mitochondrial DNA markers support species status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae)

Summary. Spring queens of Bombus cryptarum and B. magnus from 2 localities in Brandenburg/Germany and Scotland/United Kingdom respectively were determined by morphological characteristics. The lateral border of the collare at the border of the pronotallobus or at the episternum proved to be an especially useful character. Artificial colonies were reared from safely determined spring queens and the cephalic part of the labial glands of males from these colonies were investigated by GC/MS. The investigation identified approximately 50 compounds, as a mixture of straight chain fatty acid derivatives (alcohols, esters and hydrocarbons). The labial secretions of B. cryptarum and B. magnus are significantly different. Mitochondrial cytochrome oxidase 1 (CO1) of two queens from each locality and species were sequenced. Each species from the different localities formed a cluster. Sequence divergence between B. cryptarum and B. magnus was about 30 base substitutions and approximately 0.04 in Tamura-Nei genetic distance. Bombus cryptarum and B. magnus were closer to each other than to B. lucorum and made the sister group in the topology of the tree. Both the CO1 sequences and the labial gland secretions of males of B. cryptarum from Brandenburg and of males from artificial colonies reared from safely determined spring queens from Scotland are identical. B. cryptarum has thus, for the first time, been identified as part of the British bumble bee fauna. The differences of both the labial gland secretions, used as species recognition signals, and the genetic differences established by sequencing CO1 confirm the morphological findings that B. cryptarum and B. magnus are distinct taxa which should be treated as distinct species.Received 25 March 2004; revised 13 June 2004; accepted 15 June 2004.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.     

KKKKPLFGLFFGLF: A cationic peptide designed to exert antibacterial activity

With 14 residues organized as two domains linked by a single proline, the de novo peptide called K4 was designed, using Antimicrobial Peptide Database, to exert antibacterial activity. The N-terminal domain is composed of four lysines enhancing membrane interactions, and the C-terminal domain is putatively folded into a hydrophobic α-helix. Following the synthesis, the purification and the structural checking, antibacterial assays revealed a strong activity against gram-positive and gram-negative bacteria including human pathogenic bacteria such as Staphylococcus aureus and some marine bacteria of the genus Vibrio. Scanning electron microscopy of Escherichia coli confirmed that K4 lyses bacterial cells. The cytotoxicity was tested against rabbit erythrocytes and chinese hamster ovary cells (CHO-K1). These tests revealed that K4 is non-toxic to mammalian cells for bacteriolytic concentrations. The peptide K4 could be a valuable candidate for future therapeutic applications.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

茶树硝酸还原酶基因克隆及表达分析

利用茶树全器官转录组文库中硝酸还原酶(NR)的EST,通过RACE技术扩增出NR基因的cDNA,并利用实时荧光定量PCR检测了NR基因在不同茶树品种中的表达。结果表明:NR基因cDNA全长2 927bp,开放阅读框2 652bp,编码一个有884个氨基酸蛋白质,GenBank登录号为JX987133。经BlastX比对,与GenBank中登录的烟草NR相似性达到74%。茶树NR蛋白属于亲水性蛋白,可能为胞质蛋白。25个茶树品种叶片中NR表达水平差异明显,最高值是最低值的22.75倍。因NR是植物氮代谢过程中的关键限速酶,推测25个茶树品种间氮吸收利用能力存在差异。