Cloning and Expression of Gonadotrophin Releasing Hormone Receptor in Apis cerana cerana

[Objective] This study was to clone the GnRH （gonadotropin-releasing hormone）, and to investigate its expression in Apis cerana cerana. [Method] The cDNA sequence of GnRHR gene was amplified from Apis cerana cerana by using RT-PCR techniques. It was conducted with bioinformatics analysis and the in situ hybridization histochemistry of its expression products was studied. [Result] The sequence analy- sis showed that the full cDNA sequence was 1 050 bp with the open reading frame of 1 050 bp, and it encoded 349 amino acid residues. The deduced amino sequence included 7 transmembrane regions, and the predicted molecular mass and isoelectric point were 40.6 kD and 9.54, respectively. The cluster analysis showed that the GnRHR from ＇,4. cerana cerana had close relationship to the GnRHR II from other insects. In situ hybridization showed that Bee-GnRHR staining was specifically localized to the brain, intestine, fat body and testis. [Conclusion] The results indicated that the GnRHR provided molecular bond for the reproduction and metabolism for insects, and suggested a functional role for bee-GnRHR signaling in the coupling of reproduction activities and environment conditions.

Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

By screening the worker （Apis cerana cerana） heads cDNA library using a fragment of the mrjp3 gene of Apis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 （AccMRJP3） cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly （A） tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame （ORF） with 1 779 bp encoding 593 amino acids. The un-translated regions （UTR） of the 5′ end and 3′end are 46 bp and 160 bp in length, respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

Role of c-Jun NH_(2)-terminal kinase-mediated mitogen-activated protein kinase pathway in response to pesticides in Apis cerana cerana

The mitogen-activated protein kinase(MAPK)cascade pathway plays an important role in regulating stress responses.The function of the c-Jun NH_(2)-terminal kinase(JNK),a component of the MAPK cascade pathway,in Apis cerana cerana(Acc)remains unclear.Here,JNK was isolated and identified from Acc.Bioinformatics analyses revealed there is a typical serine/threonine protein kinase catalytic domain in the AccJNK protein.An expression profile analysis showed that AccJNK was significantly induced by pesticide treatments.To further explore the functional mechanisms of AccJNK,a yeast 2-hybrid screen was performed,activator protein-1(AP-1)was screened as the interaction partner of AccJNK,and the interaction relationship was further verified by pull-down assay.Quantitative real-time polymerase chain reaction showed the expression pattern of AccAP-I was similar to that of AccJNK.After a knockdown of AccJNK or AccAP-I by RNA interference,the survival rate of Acc after pesticide treatments increased.Additionally,the expression levels of antioxidant-related genes and the activities of antioxidant enzymes increased,suggesting that the knockdown of AccJNK or AccAP-I increased the antioxidant capacity of bees.Our study revealed that the JNK-mediated MAPK pathway responds to pesticide stress by altering the antioxidant capacity of Acc.

Expression of a bee venom phospholipase A_2 from Apis cerana cerana in the baculovirus-insect cell

Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2) gene from the Chinese honeybee(Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector.Tn-5B-4(Tn) cells were transfected with the recombinant bacmid DNA for expression.Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa.Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins.The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2(BvPLA2 of the European honeybee,Apis mellifera) polyclonal serum.The reaction resulted in a double glycosylation band,which agrees with the band generated by the native AmPLA2 in Western blot analysis.The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg).In summary,the recombinant AccPLA2 protein,a native BvPLA2-like structure with corresponding biological activities,can be glycosylated in Tn cells.These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.

Insulin receptor participates in the peripheral olfactory processes of honey bees (Apis cerana cerana)

Insulin receptors(InR)are an integral component of the insulin/insulin-like growth factor signaling pathway,which plays a vital role in insect development,lifespan,reproduction,and olfactory sensitivity.However,whether InR participate in the periph-eral olfactory system of insects remains unclear.Recently,we found that 2-heptanone(2-HT)affects AcerlnR expression,the gene for an InR protein,in Apis cerana cerana.We then examined the spatiotemporal expression profile of the gene in A.cerana cerana.The mRNA of AcerlnR was primarily expressed in the antennae,wings,and legs of forager bees,which are probable chemosensory tissues.The results of fluorescence competitive binding assays,combined with site-directed mutagenesis,demonstrated that AcerOBP6 and AcerOBP14 exhibit strong binding affinities to 2-HT.Furthermore,after foragers were fed with double-stranded AcerlnR,the expression levels of AcerOBP6 and AcerOBP14 decreased significantly,as did the electroantennogram responsiveness to 2-HT and some other odorants.In conclusion,our findings provide a foundation for understanding the in-volvement of AcerlnR in the odor perception of A.cerana cerana.Moreover,they offer novel insights into the olfactory recognition mechanism in insects.

中华蜜蜂养殖业在我国的发展方向和解决方案

分析中华蜜蜂养殖规模小和多点性特点,提出解决我国中华蜜蜂养殖业的方案。建议采用活节叠加式仿生蜂箱,使养蜂技术标准化和简单化、门槛降低,可扩大养殖的规模;使用网络技术、大数据分析技术实现养殖的精准管理;最终在国家政策的引领下,实现中华蜜蜂产业集团化。

重庆彭水县中华蜜蜂形态指标测定及比较研究

采集重庆彭水县中华蜜蜂(Apis cerana cerana)主要栖息区内18个乡镇的蜜蜂样本,对工蜂吻长、前翅长等6项主要形态指标进行测定,并将彭水县中华蜜蜂形态指标数据与湖北、江西等17地已报道的中华蜜蜂形态指标进行比较。结果显示,彭水县中华蜜蜂的翅长、翅宽、T3+T4背板长平均值处于中上水平,暗示其采集能力和花蜜储存能力有一定优势。试验结果可为当地中华蜜蜂遗传资源调查与保护提供形态学数据参考。