【作者】 巩志宏；

【导师】 胥保华；

【作者基本信息】 山东农业大学 ， 动物营养与饲料科学， 2011， 硕士

【摘要】 蜜蜂的寿命及抗性与蜜蜂的抗氧化能力有直接联系,生物体内活性氧（ROS）的积累能引起DNA、蛋白质等生物大分子的氧化性损伤,如甲硫氨酸被氧化而变为甲硫氨酸亚砜,可导致其所在蛋白的生物学活性降低或丧失。生物体内普遍存在的甲硫氨酸亚砜还原酶（methionine sulfoxide reductases, Msrs）可催化甲硫氨酸亚砜还原为甲硫氨酸,使其所在蛋白质恢复活性。甲硫氨酸亚砜还原酶的这种催化还原作用,在蛋白修复及间接抗氧化方面发挥着重要的作用。目前,关于Msrs的研究主要集中在生物抗氧化及寿命调节方面,在蜜蜂中的研究很少。蜜蜂做为一种授粉昆虫,在农业生产及维持生态平衡中起重要作用。蜜蜂外出采蜜时会遭受到多种环境胁迫,为明确MsrA在蜜蜂抗氧化及发育中的作用。本研究以中华蜜蜂（Apis cerana cerana）为材料,首次从中华蜜蜂中克隆得到甲硫氨酸亚砜还原酶A（AccMsrA）基因,并利用实时荧光定量PCR（qRT-PCR）及免疫组化的方法,首次分析了AccMsrA在昆虫不同发育阶段转录水平的变化,探索了其在紫外线（30 mj/cm²）、高温（43℃）及H₂O₂（2 mM）三种不同氧化刺激下的表达变化,为进一步研究该基因的功能及作用机理奠定了基础。主要研究结果如下:1、通过RT-PCR和RACE-PCR的方法从中华蜜蜂中克隆得到MsrA基因,命名为AccMsrA,其GenBank注册号为HQ219724,cDNA全长1,540 bp,包含136 bp的5′UTR,753 bp的3′UTR和651 bp的ORF,编码一个有217个氨基酸残基,分子量约为25 kD的蛋白。同源分析得出AccMsrA同来源于埃及斑蚊、榕果蝇、致倦库蚊中的几种MsrA的同源性分别为49.6%、45.8%和51.3%。序列分析发现,AccMsrA具有MsrA的保守区,该保守区内存在典型特征序列GCFW,其中的半胱氨酸残基是其催化还原过程中的一个活性位点。cDNA序列与基因组序列比对后发现AccMsrA基因组序列内存在四个内含子,其中一个内含子位于5′UTR内。2、通过iPCR的方法获得了1,364 bp的AccMsrA基因启动子序列,经软件分析发现,该区域中存在tramtrack （Ttk）、cell factor 2-II （CF2-II）、hunchback （Hb）、Broad-Complex （BR-C）四种与发育相关的顺式作用元件,还有五个heat shock factor （HSF）热激响应元件。qRT-PCR检测到AccMsrA在五日龄幼虫及成虫头部具有较高表达。推测AccMsrA参与了蜜蜂的生长发育调节并在成虫不同组织部位存在表达差异。此外,AccMsrA的转录水平受紫外线（30 mj/cm²）、高温（43℃）和H₂O₂（2 mM）的诱导而提高,表明其在抵抗外界氧化刺激过程中发挥重要的作用。3、分别以pMD18-T simple和pET-30a（+）为克隆载体和表达载体构建了AccMsrA基因编码区的原核融合表达载体AccMrsA-pET-30a（+）,转化大肠杆菌BL21（DE3）并对其表达条件进行优化,在IPTG诱导下表达出大小约为29 kDa的融合蛋白。大量诱导表达目的蛋白,经纯化后免疫小鼠,采集含多克隆抗体的血清,通过Western blot鉴定其具有特异性并测定得出其效价为1:1000。4、通过对五日龄蜜蜂幼虫的免疫组化定位得出AccMsrA在五日领幼虫体内主要存在于腹部及肠壁,进一步推测AccMsrA可能参与蜜蜂从幼虫到蛹变态发育过程中腹部器官的发育调节。更多还原

【Abstract】 ROS generated by stimulation lowers the cellular antioxidant capacity, alters protein function and damages proteins. Methionine can be oxidized to methionine sulfoxide, which can lead to reduced biological activity of proteins which or loss. Methionine sulfoxide reductases （Msrs） catalyze the reduction of methionine sulfoxide （MetO） to methionine and play key roles in protein repair and reactive oxygen species （ROS） scavenging. Previous studies of MsrAs have mainly focused on antioxidant defenses and modulation of the aging process, and little information about MsrA in bees has been available until now. Here, a novel MsrA gene designated AccMsrA was isolated from Apis cerana cerana. Quantitative real-time PCR （qRT-PCR） and Immunohistochemical localization were used study the AccMsrA mRNA expression in different developmental stages and tissues. Furthermore, the accumulation of AccMsrA mRNA was induced by oxidative stimulation . The results as follow:1. Based on the conserved region of insect MsrA genes, primers were designed, and then RT-PCR and RACE-PCR were performed to amplify the region of AccMsrA. The full-length cDNA of AccMsrA （GenBank accession no. HQ219724） is 1,540 bp, and it contains a 136-bp 5′untranslated region （UTR） and a 753-bp 3′UTR. The cDNA harbors a 651-bp open reading frame （ORF） encoding a protein of 217 amino acids. The theoretical molecular mass of AccMsrA was calculated as approximately 25.0 kDa. Sequence alignment analysis showed that AccMsrA shares high similarity with other insect MsrAs. A multiple sequence alignment showed that AccMsrA shares 49.6%, 45.8% and 51.3% identity with the MsrAs from Aedes aegypti, Drosophila erecta and Culex quinquefasciatus, respectively. The highly conserved region GCFW, which is the fingerprint of MsrAs, was found in the N-terminus. Moreover, a cysteine residue was identified in the conserved GCFW region that is involved in the catalytic mechanism. 2. Analysis of the 5′flanking region of AccMsrA revealed a group of putative cis-acting elements that are related to the regulation of development and responses to external stimuli. Quantitative real-time PCR （qRT-PCR） detected high levels of AccMsrA mRNA during pupation （fifth-day larvae） and in the head tissue of adults. Immunohistochemical localization showed that AccMsrA was primarily concentrated in the abdomen and intestinal tract wall in fifth-instar larva. Furthermore, the expression of AccMsrA was up-regulated by multiple oxidative stresses, including ultraviolet （UV）-light （30 mj/cm²）, heat （43℃） and H₂O₂ （2 mM）. These results indicate that AccMsrA might fulfill an important role in the regulation of insect development and in response to various environmental stresses.3. The complete ORF sequences of AccMsrA were cloned into the pET30a （+） expression vector. The recombinant expression vectors were transformed into E. coli strain BL21 （DE3） and then induced. The 29 KDa recombinant proteins were separated and purified. Antisera were produced by injecting purified recombinant AccMsrA protein into white mice.4. The Immunohistochemical localization results revealed that AccMsrA was widely distributed, and high activity was detected in the abdomen and the intestinal tract wall, in fifth-day larvae. This result implies that AccMsrA might be involved in the the growth of segmental ventral organs in pupae.更多还原