意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam对靶基因USP和P300的表达调控

【目的】本研究旨在通过饲喂法对意大利蜜蜂Apis mellifera ligustica工蜂幼虫肠道中ame-miR-bantam进行过表达与敲减,明确ame-miR-bantam对靶基因USP和P300表达的调控作用。【方法】通过饲喂ame-miR-bantam模拟物(mimic-ame-miR-bantam)和抑制物(inhibitor-ame-miR-bantam)及相应的阴性对照mimic-NC-ame-miR-bantam和inhibitor-NC-ame-miR-bantam,对意大利蜜蜂工蜂4-6日龄幼虫肠道组织中的ame-miR-bantam分别进行过表达和敲减。利用生物信息学软件进行ame-miR-bantam的靶向预测及分析。通过RT-qPCR检测4-6日龄意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam的过表达和敲减效果及靶基因USP和P300的相对表达量。【结果】相较于饲喂mimic-NC-ame-miR-bantam,饲喂mimic-ame-miR-bantam后,ame-miR-bantam在意大利蜜蜂4-6日龄工蜂幼虫肠道中均显著上调;相较于饲喂inhibitor-NC-ame-miR-bantam,饲喂inhibitor-ame-miR-bantam后,ame-miR-bantam在4日龄工蜂幼虫肠道中显著下调,在5日龄和6日龄工蜂幼虫肠道中下调表达。ame-miR-bantam共靶向222个基因,可注释到细胞进程等35个GO条目和Wnt信号通路等160条KEGG通路。相比于饲喂mimic-NC-ame-miR-bantam的阴性对照组,过表达ame-miR-bantam后,4日龄工蜂幼虫肠道中USP和P300的表达量均为上调;5日龄工蜂幼虫肠道中USP的表达量下调,P300显著下调表达;6日龄工蜂幼虫肠道中USP和P300均显著下调表达。相比于饲喂inhibitor-NC-ame-miR-bantam的阴性对照组,敲减ame-miR-bantam后,4和5日龄工蜂幼虫肠道中USP和P300的表达量均为上调;6日龄工蜂幼虫肠道中USP显著上调表达,P300的表达量上调。【结论】通过饲喂模拟物和抑制物能够分别实现意大利蜜蜂工蜂幼虫肠道中ame-miR-bantam的有效过表达和敲减;幼虫肠道中ame-miR-bantam可负调控USP和P300的表达。

西方蜜蜂几丁质合成酶基因的生物信息学分析及功能研究

【目的】本研究旨在明确西方蜜蜂Apis mellifera几丁质合成酶(chitin synthase,CHS)基因的分子特性,并揭示CHS在西方蜜蜂工蜂幼虫响应蜜蜂球囊菌Ascosphaera apis胁迫的免疫应答中的功能。【方法】利用相关生物信息学软件预测和分析西方蜜蜂CHS蛋白的分子特性、保守基序和结构域。使用MEGA X软件对西方蜜蜂和其他昆虫CHSs的氨基酸序列进行系统进化分析。采用体外转录法合成CHS和egfp的dsRNA,饲喂蜜蜂球囊菌胁迫的西方蜜蜂工蜂3日龄幼虫以进行RNAi,并利用RT-qPCR检测西方蜜蜂工蜂5日龄幼虫肠道中CHS及宿主响应蜜蜂球囊菌侵染的免疫相关基因abaecin,apidaecin,birc5,defensin-1和PGRP-S2的表达量。【结果】西方蜜蜂CHS蛋白含有1572个氨基酸,属于20种氨基酸,其中正电荷氨基酸与负电荷氨基酸分别为177和169个,分子量约为178.77 kD,等电点为6.65;含纤维素合酶CESA3催化结构域。在西方蜜蜂和东方蜜蜂A.cerana等共13种昆虫的CHSs中均鉴定到3个Motif(Motif 1,Motif 2和Motif 3)和2个结构域(Chitin_synth_2和Glyco_trans_2_3)。系统进化树表明,膜翅目的西方蜜蜂与东方蜜蜂的CHSs的氨基酸序列一致性最高,聚为一支且自举值为100。相较于ds egfp饲喂对照组,ds CHS饲喂组5日龄幼虫肠道中CHS的表达量显著下调,干扰效率为29.40%,abaecin,birc5和defensin-1的表达量均极显著上调,PGRP-S2的表达量显著上调,apidaecin的表达量极显著下调。【结论】西方蜜蜂CHS可能是胞内亲水性跨膜蛋白,西方蜜蜂与其他昆虫CHSs的氨基酸序列具有较高的保守性,其中与东方蜜蜂的CHS的氨基酸序列之间保守性最高,CHS影响西方蜜蜂工蜂幼虫响应蜜蜂球囊菌胁迫的免疫应答。

Formation of Carbon Nitride Films by Nitrogen Ion Bombardment on C_(60) Films

The synthesis of covalent carbon nitride films becomes an important subject in the materials research field.As a new synthetic method two low-energy(400 and 1000eV)nitrogen ion beams are used to bombard on C_(60) thin films individually.The bombarded films are used for Raman and x-ray photoelectron spectroscopy(XPS)measurements.The results of the analyses show that under the bombardment of 400eV nitrogen ion beam,the film still contains a large amount of undestructed C_(60) molecules.In the case of l000eV bombardment,only a little amount of C_(60) molecules is kept undestructed.The experimental results also show that the destructed carbon species will combine chemically with nitrogen ions to form stable covalent carbon nitride,confirmed by the Raman peaks of,e.g.,2240cm-1.The XPS Nls and Cls lines also indicate the formation of covalent carbon nitride in the bombarded films.

Ion-Beam Bombarding Effects on Deposition of Carbon Nitride Films by Laser Ablation

In order to obtain high quality carbon nitride films,x-ray photoelectron spectroscopy and Raman spectrum were used to analyze several kinds of film:(a)laser-ablated amorphous carbon(a-C)films,(b)a-C films after a 1 keV nitrbgen ion beam bombardment.(c)carbon nitride(CN_(x))films synthesized by ablation assisted with ion beam bombardment,(d)CN_(x) films after a 50eV nitrogen ion beam bombardment,and(e)CN_(x) films after a 1 keV nitrogen ion beam bombardment.The comparison among these films showed that the concurrent deposition of CN_(x) films by ablation and a nitrogen ion beam should be better than the post-treatment of a-C films by nitrogen ion beam bombardment.In the case of concurrent deposited CN_(x) films,a post-treatment by a nitrogen ion beam bombardment with an appropriate energy(possibly less than 1 keV)was proposed to improve the ratio of C-N binding structures in the deposited films.