Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the...Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.展开更多
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 ce...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.展开更多
Despite the urgent need for conservation consideration,strategic action plans for the preservation of the Asian honeybee,Apis cerana Fabricius,1793,remain lacking.Both the convergent and divergent adaptations of this ...Despite the urgent need for conservation consideration,strategic action plans for the preservation of the Asian honeybee,Apis cerana Fabricius,1793,remain lacking.Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy.Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts,as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities.Here,we investigated genome variations in 362 worker bees representing almost all populations of mainland A.cerana to understand how evolution has shaped its population structure.Whole-genome single nucleotide polymorphisms(SNPs)based on nuclear sequences revealed eight putative subspecies,with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies.Our results demonstrated that most classic morphological traits,including body size,were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism.Thus,such morphological traits were not suitable for subspecific delineation.Conversely,wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes.Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor.Based on our findings,we propose that criteria for subspecies delineation should be based on evolutionary independence,trait distinction,and geographic isolation.We formally defined and described eight subspecies of mainland A.cerana.Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units,guiding colony introduction and breeding.展开更多
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...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.展开更多
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）...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.展开更多
Within a honeybee population, due to polyandry, there are super-sister and half-sister relations, thus many sub-families exist. For the Chinese honeybee (Apis cerana cerana F.) a phylogenetic dendrogram has been const...Within a honeybee population, due to polyandry, there are super-sister and half-sister relations, thus many sub-families exist. For the Chinese honeybee (Apis cerana cerana F.) a phylogenetic dendrogram has been constructed in which 4 sub-families are clustered based on DNA fingerprint patterns. And it has been observed that each kind of workers is distributed to several different sub-families.展开更多
[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 ...[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.展开更多
基金supported by the National Basic Research Program of China (2007CB109203 and 2009CB118902)
文摘Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.
基金supported by the Shandong Provincial Natural Science Foundation(No.ZR2019MC050)special funds for the Efficient Ecological Agriculture Innovation Project of the Taishan Industry Leading Talent Program(No.LJNY202003)the earmarked fund for the China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs(No.CARS-44).
文摘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.
基金supported by the National Natural Science Foundation(NSF)of China(32270475)Program of Ministry of Science and Technology of China(2018FY100403)+3 种基金National Special Support Program for High-level Talents(Ten-Thousand Talents Program)2115 Talent Development Program of China Agricultural University through Xin Z.S.L.is supported by Funds for International Cooperation and Exchange of the National Natural Science Foundation of China(3211001043)supported by the NSF of China(31470123)Jilin Science and Technology Program(20030561)through X.L.S.H.P.is supported by the National Mission on Himalayan Studies(NMHS)-Almora,Ministry of Environment,Forest and Climate Change,Government of India,through grant GBPNI/NMHS-2017-18/MG-12。
文摘Despite the urgent need for conservation consideration,strategic action plans for the preservation of the Asian honeybee,Apis cerana Fabricius,1793,remain lacking.Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy.Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts,as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities.Here,we investigated genome variations in 362 worker bees representing almost all populations of mainland A.cerana to understand how evolution has shaped its population structure.Whole-genome single nucleotide polymorphisms(SNPs)based on nuclear sequences revealed eight putative subspecies,with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies.Our results demonstrated that most classic morphological traits,including body size,were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism.Thus,such morphological traits were not suitable for subspecific delineation.Conversely,wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes.Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor.Based on our findings,we propose that criteria for subspecies delineation should be based on evolutionary independence,trait distinction,and geographic isolation.We formally defined and described eight subspecies of mainland A.cerana.Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units,guiding colony introduction and breeding.
基金Project (No 2007AA10Z324) supported by the National High-Tech Research and Development Program (863) of China
文摘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.
基金the National Natural Science Foundation of China(30200206) Zhejiang Provincial Natural Science Foundation of China(302113).
文摘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.
文摘Within a honeybee population, due to polyandry, there are super-sister and half-sister relations, thus many sub-families exist. For the Chinese honeybee (Apis cerana cerana F.) a phylogenetic dendrogram has been constructed in which 4 sub-families are clustered based on DNA fingerprint patterns. And it has been observed that each kind of workers is distributed to several different sub-families.
基金Supported by the Science and Technology Planning Project of the Education Department of Shaanxi Province(11JK0618)~~
文摘[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.
