【作者】 邹勇；

【导师】 夏庆友； 赵萍；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 属于鳞翅目昆虫的家蚕,体内物质的代谢的重要器官—马氏管与其他昆虫的马氏管(肾管)乃至哺乳动物的肾脏有着很大的相关性,也具有其独特的特征。对于家蚕马氏管重要功能性蛋白质的发现和研究可以为我们更好地理解昆虫马氏管(肾管)的功能,在漫长的进化过程中,因为不同的进化需要,昆虫和哺乳动物共有和不同的进化结果。另外,作为能够大量吐丝结茧的昆虫,家蚕的巨大经济价值已长久被人们所关注。代谢作为消化过程后的必要环节,其与家蚕生长发育的调节作用不容忽视。因此,对于家蚕马氏管的研究对于提高家蚕体质,进而提高产丝效率和产丝量可以起到一个很好推动作用。而且,家蚕马氏管具有显著区段特征的特殊构造对于研究马氏管(肾管)不同区段的功能提供了很好的条件。本研究利用丙烯酰胺凝胶电泳(SDS-PAGE)结合液相质谱(LC MS/MS)、双向电泳(2D)结合基质辅助激光解析质谱(2D-MALDI-TOF/MS)的方法分析了家蚕五龄第5天马氏管的主要表达蛋白质；并对五龄第5天的各段马氏管、五龄第5天与化蛹第1天整段马氏管的表达蛋白质进行了比较；还对蛾期马氏管代谢产物中存在的主要蛋白质进行了鉴定；利用家蚕的基因组序列、表达序列标签(ESTs)数据以及基因芯片数据,对具有显著时期和区段分布特点的蛋白质BmAGXT2、BmHYI、Bmhibadh的编码基因Bmagxt2、Bmhyi、 Bmhibadh进行了克隆和分析；同时采用RT-PCR、原核表达、Western blotting等技术对Bmagxt2、Bmhyi的功能进行了研究；测定了BmAGXT2在马氏管五龄期到蛹期、五龄期各区段中的酶活力。1.家蚕马氏管五龄第5天LC MS/MS及2D-MALDI-TOF MS分析LC MS/MS液向-质谱联用鉴定复杂蛋白质混合物的技术现在已广泛应用在蛋白质组学平台中。在本研究中采用了LTQ VELOS质谱仪,成功的鉴定了家蚕五龄第5天马氏管表达蛋白质。我们采用了构建正反库的方式和TPP软件的筛选功能对鉴定结果进行了质量控制,增加了蛋白的鉴定可信度。最终,我们一共鉴定到了1367个非重复蛋白质,其中80.54%的鉴定蛋白质理论分子量分布在10-100kD之间,98.90%的鉴定蛋白质的理论等电点分布在4-12之间；而家蚕9倍基因组预测的所有蛋白质中有82.48%的理论分子量分布在10-100kD之间,97.82%的理论等电点分布在4-12之间。其中有41个蛋白质同时在双向电泳中检测到,且这些蛋白质在双向电泳图谱中发现的表达量和APEX分析结果相似。我们对这些蛋白质进行了家族分类、GO分类、pathway分类后发现：家蚕的马氏管不仅完成了调节体内水分和盐分的作用,除了功能相似之外,其与哺乳动物的肾脏在多个代谢通路上有着相似之处,并且二者可能还有更多的关联。作为专一食桑叶的昆虫,家蚕选择了以马氏管作为场所来完成对桑叶挥发油成分的代谢。在马氏管中的GSTs、P450s、乙醇脱氢酶(alcohol dehydrogenases)等蛋白质家族来处理外源性和内源性的有害物质,达到机体免受其伤害的目的。2.五龄第5天马氏管分段双向电泳与质谱分析及代谢产物中蛋白质的鉴定通过颜色观察和查阅文献,我们发现家蚕马氏管至少可以分为3个区段,对这三个区段分别进行DAPI染色30min后的结果显示,第Ⅰ区段染色主要集中在马氏管管腔内侧；第Ⅱ区段染色质呈网状分布；第Ⅲ区段染色质呈颗粒状分布。说明这3个区段有较大的区别。为了了解马氏管各区段的蛋白质分布情况,分别对这3个区段进行了双向电泳,我们发现BmAGXT2、 BmHYI、Bmhibadh、BmActin3在各区段中有较大差异,其中BmAGXT2主要分布于第1区段(远离直肠端),第2区段、第2区段中分布较少；BmHYI、Bmhibadh在第1区段的分布量也大于其他区段；而BmActin3在第3区段中的量最大。在研究中,我们对化蛾第2天马氏管内容物中的蛋白质情况进行了双向电泳-质谱分析,成功鉴定到其中3个蛋白点,均为典型的抗菌肽蛋白质hemolin,推测可能蛹期及蛾期免疫有关。3.家蚕幼虫期和蛹期马氏管差异蛋白质组学分析利用蛋白质双向电泳技术对家蚕五龄第5天及化蛹第1天的马氏管组织进行比较,并采用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)对表达量差异较大的蛋白点进行肽指纹图谱鉴定,应用GPMAW8.00软件对NCBI蛋白质数据库中所有来自于P50/Dazao的蛋白质与家蚕9倍基因组预测蛋白质库共同构建本地数据库,对试验采集到的肽指纹图谱进行分析。共检测到360～430个蛋白点,主要分布在分子质量15～80kD、等电点4.0～9.5的范围内。两个时期共有17个表达量有显著差异的蛋白质被成功鉴定,其中包括与能量代谢相关蛋白质、热激蛋白、V型H+ATP合酶、30K蛋白以及与肾脏功能密切相关的丙氨酸-乙醛酸氨基转移酶2(BmAGXT2),3一羟基异丁酸脱氢酶(Bmhibadh)等。研究结果可以为家蚕马氏管怎样实现从幼虫期代谢桑叶消化产物功能为主到蛹期代谢消耗脂肪体及储藏蛋白的产物功能为主的巨大转变提供蛋白质水平上的依据。4. Bmagxt2、Bmhyi、Bmhibadh基因的克隆、序列分析及mRNA表达根据基因组中Bmagxt2、Bmhyi、Bmhibadh的序列,设计引物,克隆基因,经过酶切和测序验证得到三个基因的阳性克隆。从基因结构上分析, Bmagxt2基因CDS长为2080bp,编码的蛋白含260个氨基酸残基,预测蛋白的分子量大小为50.38kD,等电点为7.70,具有9个外显子,8个内含子,在基因组上仅有一个拷贝,位于11号染色体nscaf3031上。SignalP预测没有信号肽,为分泌型蛋白；TMHMM预测没有跨膜结构。Bmhyi基因CDS长为783bp,编码的蛋白含260个氨基酸,预测蛋白的分子量大小为29.17kD,等电点为6.10,含有4个外显子,3个内含子,在基因组上仅有一个拷贝,位于nscaf2360上,染色体定位情况未知。SignalP预测没有信号肽,为分泌型蛋白；TMHMM预测没有跨膜结构。Bmhibadh基因CDS长为969bp,编码的蛋白含322个氨基酸残基,预测蛋白的分子量大小为34.01kD,等电点为9.24,含有6个外显子,5个内含子,在基因组上仅有一个拷贝,位于17号染色体nscaf2865上。SignalP预测信号肽为第1-21个氨基酸残基(MAARTILSTQCLYTAARRAYS); TMHMM预测前60个氨基酸残基为跨膜结构。为了了解三个基因在家蚕各时期、组织的表达特征,我们利用家蚕ESTs、基因芯片数据以及RT-PCR方法进行了分析。ESTs分析结果显示Bmagxt2、Bmhyi、Bmhibadh均具有EST证据。在家蚕马氏管、中肠中Bmagxt2有EST证据。在家蚕卵巢、血细胞、马氏管、中肠、脂肪体、丝腺中Bmhyi有EST证据,在家蚕中肠、翅原基、马氏管、胚胎、血细胞、脂肪体、丝腺中Bmhibadh有EST证据。对家蚕全基因组芯片的组织表达数据的分析结果显示：Bmagxt2基因在家蚕五龄第3天的各组织中的mRNA的量都很低,除卵巢、精巢、脂肪体这三个组织有极少量外,其他组织中几乎没有mRNA的存在。Bmhyi基因在丝腺、卵巢、精巢、马氏管、体壁、头、血液、脂肪体、中肠中均有不同程度的表达,其中丝腺中表达量较低,而马氏管、血液、脂肪体、中肠中表达量较高,尤其马氏管中表达量很高。Bmhibadh基因在在家蚕五龄第3天的各组织中几乎没有表达。对早期胚胎和熟蚕期到蛾期的芯片数据的分析结果显示：Bmhibadh发育时期、早期胚胎无数据。Bmagxt2基因在胚胎早期和上蔟第8-9天有很微弱量的表达,其他时期几乎不表达；Bmhyi基因在整个胚胎期、熟蚕期至蛾期都没有表达。为了进一步补充和验证表达谱,我们进行了半定量RT-PCR检测。结果显示：Bmagxt2基因在家蚕五龄第3天幼虫生殖腺、体壁、脂肪体表达量较高,在头和马氏管中有微弱表达；而从1龄眠期到五龄第7天的期间内均有表达。Bmhyi基因在家蚕五龄第3天幼虫脂肪体、表皮、血细胞中有微弱表达,马氏管中表达量较高；而从蚁蚕期到上蔟后第10天期间都有表达,整体表现为幼虫期表达量大于蛹期,上蔟后36-72h表达量小于上蔟后96h到上蔟后第10天。Bmhibadh基因在家蚕五龄第3天幼虫各组织中均有表达,马氏管和丝腺中的表达量略高于其他组织；而在幼虫期各时间点均有一定量表达,总体趋势表现为眠期表达量小于起蚕期；上蔟后该基因几乎无表达,一直到上蔟后第8天才有极微弱的表达量,并一直保持到蛾期。5. Bmagxt2、Bmhyi的原核表达、抗体制备及western blotting检测为了对基因功能进行进一步研究,我们将Bmagxt2和Bmhyi分别与p29和p28表达载体相连接,相应命名为Bmagxt2/p29和Bmhyip28,并转化到BL21(DE3)表达菌株中。IPTG诱导表达后分别得到分子量约为51kD和30kD的重组包涵体蛋白质,而目标蛋白质分子量约为50.38kD和29.17kD,加上6个组氨酸标签,大小约为51kD和30kD,与预测分子量大小基本一致。通过Ni+亲和柱和电洗脱纯化,得到了较高浓度的原核表达包涵体蛋白质BmAGXT2. BmHYI。将这两个蛋白质免疫成年健康雄兔后,取得血清,纯化得到多克隆抗体。利用抗体检测Bmagxt2和Bmhyi在家蚕五龄第5天各组织的表达情况,结果显示,BmAGXT2蛋白质在马氏管和血液中有表达,尤其在马氏管中表达量很高,这与我们在双向电泳试验中发现的现象一致。Bmhyi蛋白质只在马氏管中有表达。而进一步的双向western blotting结果显示,与五龄第5天相比较,化蛹第1天马氏管中的BmAGXT2和BmHYI都有很大程度的减少,这一结果之前的双向电泳中观察的结果一致。6.丙氨酸-乙醛酸氨基转移酶2(BmAGXT2)活性检测为了研究BmAGXT2蛋白在马氏管中的活性情况是否与其表达量具有相关性,我们参照其在人和小鼠同源蛋白的酶活性测定方法,测定了BmAGXT2从五龄第3天到化蛹第7天以及其在五龄第5天马氏管各区段中的酶活性,结果显示：(1)BmAGXT2在家蚕幼虫马氏管中的活性从五龄第3天到五龄第7天逐渐升高；之后,则逐渐降低,整个蛹期,其活性在一个比较稳定的水平。(2)BmAGXT2在幼虫期马氏管各段中的活性以第一区段(离结肠接口最远段)最高,第二区段次之,第三区段(靠近结肠接口段)最小。暗示BmAGXT2可能与代谢强度及蚕进食桑叶有着一定的相关性,BmAGXT2主要在家蚕马氏管的第一区段中发挥其作用。更多还原

【Abstract】 Silkworm(Bombyx mori) belongs to the lepidopteran insect. Its malpighian tubules, the important organs for metabolism in the body, have not only the distinctive characteristics but also great relevance to the renal tube of other insects and even the kidney tube of mammals. The discovery and research, for significant functional proteins of the silkworm(Bombyx mori), will be favor of comprehending the function of malpighian tubules in insects. Moreover, it is helpful to find out the similar and different evolution results between the insects and the mammals, because of the different evolution requirements during long evolutionary process. What’s more, so far, the economic value of Bombyx mori for silk has been paid long attention to. Because Metabolism plays the necessary role after the process of digestion, and it has the regulatory impact on the growth and development of silkworm. Hence, the research on malpighian tubules of silkworm will make contributions to enhancing the silkworm’s physical health and impoving the the efficiency of silk production. Furthermore, fortunately, the malpighian tubules of silkworm possess the typical structure with various obvious sections,which provids better opportunity to research the functions of the different sections in the malpighian tubules.In this study, we analysised the proteins of malpighian tubules on day-5fifth larve with acrylamide gel electrophoresis (SDS-PAGE)-LC MS/MS and2D-MALDI-TOF/MS technology. And compared the different expression proteins between day-5fifth larve day-1pupa as well as each section of malpighian tublues. Also the main protein present in the moth of malpighian tubules metabolites were identified; Use of the silkworm genome sequence, expression sequence tags (ESTs) data as well as gene chip data, with significant periods and the section of the distribution characteristics of protein BmAGXT2, BmHYI, Bmhibadh the encoding gene Bmagxt2, Bmhyi, Bmhibadh carried out the cloning and analysis. RT-PCR, prokaryotic expression, Western blotting, and other technology is used to study the function of BmAGXT2, Bmhyi. Finally, we measured the activity of BmAGXT2in malpighian tublues from day-3fifth instars larvae to day-7pupa and in each section on day-5fifth instars larvae.1. Proteomics analysis of malpighian tubules of day-5fifth instars larvae using LC MS/MS combined with2D-MALDI-TOF MSLC MS/MS is a method of identifying proteins in complex mixtures using a combination of high performance liquid chromatography combined with mass spectrometry. The proteins of Day-5fifth instar larvae were identified LTQ VELOS mass spectrometer. To control the confidence, the identified peptides from SEQUEST were further validated by TPP analysis. Moreover, the FDR of the identifications estimated by searching MS/MS spectra against a target-decoy database. Finally, a total of1367proteins were identified. Approximately80.54%of these proteins mostly ranged between10-100kDa, and98.90%of these proteins had pis of4-12. Thus, the proteins were identified from9×silkworm genome that82.48%of proteins had the molecular weights mostly ranged between10-100kDa, and97.82%had pIs of4-12. Forty one proteins were simultaneously identified by2-DE. These proteins are indeed known to be highly abundant of the APEX results. Through the analysis of family classification, Gene Ontology and KEGG pathway of these identified proteins, we found the malpighian tubules of the Bombyx mori have completed the responsibility regulating the balance of water and salt in body. In addition to the similarity of function and multiple metabolic pathways, they may have more association. As a specific insect eating mulberry leaves, the Bombyx mori choses the malpighian tubules as a place to complete the metabolism of volatile components of mulberry leave. With the help of GSTs, P450s, alcohol dehydrogenases and other protein families in the malpighian tubules to deal with exogenously and endogenously harmful substances, the purpose of avoiding injuries can be achieved.2.2D electrophoresis of malpighian tubules from the Day-5fifth instars larvae and its metabolite product from the Day-2moth and protein identification by mass spectrometry.Through the observation of the color of Malpighian tubules and the previous literatures, we found that at least three sections of the silkworm Malpighian tubules can be divided. The DNA staining of these three sections by DAPI for30min showed that the chromatin of the first part mainly concentrated in the inside of lumen, and the second part formed a network structure, whereas the third part was in a particle pattern, indicating that the significant differences are present in these three sections of Malpighian tubules. To further understand the protein distribution from the different sections, we performed2D electrophoresis and found that the expression of BmAGXT2, BmHYI, Bmhibadh, and BmActin3were varied. For example, Bmagxt2was mainly expressed in the first section (away from the rectum side), but with slight expression in the second and third sections. BmHYI and Bmhibadh were also enriched in the first section. However, the high expression of BmActin was in the third section. In the study, we identified proteins form the contents of malpighian tubules of Day-2silkworm moth using two-dimensional electrophoresis and mass spectrometry. There were three proteins successfully identified which was typical antimicrobial peptides-Hemolin. These results suggest that they maight be involved in the silkworm defense system of pupal and moth stages.3. Proteomic comparison on malpighian tublues between larvae stage and pupal stage of silkworm, Bombyx moriTo Get more imformation of malpighian tublues on the proteome changes between larvae stage and pupal stage and provide new evidences on the protein level at different developmental stages of malpighian tublues of silkworm, Bombyx mori. In this study, the matrix-assisted laser desorption ionization time of flight mass spectrometry were applied for identifying the different spots which had more expression of the2D map, and all the protein sequences from P50/Dazao on NCBI combined silkworm proteins database, were used to build local-database by the software GPMAW8.00. GPMAW also used to analyze peptide fingerprint masses. There were about360-430spots were obtained by silver staining from the malpighian tublues after2D-PAGE. Most of them were distributed in the area from15to80kD with pI4-9.5. We had successfully identified17markedly different proteins between larvae stage and pupal stage. They included not only the proteins in association with energy metabolism, but also heatshock proteins, Vacuolar-type H+-transporting ATPase,30K lipoprotein, and alanine-glyoxylate aminotransferase2,3-hydroxyisobutyrate dehydrogenase which have important functions in mammal kidney. The discovery and identification of these proteins can offer a valuable insights into how the malpighian tublues of silkworm adaptting the great changes on the protein level, from the larvae stage transporting the metabolic products of mulberry leaves digestion to the pupal stage transporting the metabolic products of fat body and storage proteins digestion.4. Cloning, sequence analysis and mRNA expression of Bmagxt2, Bmhyi and BmhibadhThree pairs of primers were synthesized based on the Bmagxt2, Bmhyi and Bmhibadh. Three positive clones including three genes were obtained. The CDS of Bmagxt2is2080bp, which codes260aa. Its putative molecular weight is50.38KD and pI is7.70. Seven exons and six introns are contained and only1copy exists which locates on nscaf3031of the11th chromosome on the genome. SignalP predicts that it has no signal peptide and it is secreted protein. TMHMM predicts no transmembrane structure. The CDS of Bmhyi is783bp, the proteins of which code260aa. Its putative molecular weight is29.17kD and pI is6.10. It contains4exons and3introns. Only one copy exists on the genome and locates on nscaf2360, but the condition of location chromosome is unknown. The CDS of Bmhibadh is969bp, the proteins of which code322aa. Its putative molecular weight is34.01kD and pI is9.24. It contains6exons and5introns. Only one copy exists on the genome and locates on nscaf2360of the17th chromosome. SignalP predicts that signal peptide is the1st to21th amino acid residues (MAARTILSTQCLYTAARRAYS). TMHMM predicts that the preceding60aa are transmembrane structure.In order to research the various periods of space-time expression profile of the three genes in Bombyx mori, we performed an expression analysis by using ESTs data, microarray data and RT-PCR. The ESTs analysis result shows that Bmagxt2, Bmhyi and Bmhibadh all have the EST evidences. In summary, ESTs evidences of Bmagxt2were found in silkworm malpighian tubules and in midgut. ESTs evidences of Bmhyi were found in silkworm ovary, blood cells, malpighian tubules, midgut, fat body and silkgland. ESTs evidences of Bmhibadh were found in silkworm midgut, wing primordium, malpighian tubules, embryo, blood cells, fat body and silkgland.The genome-wide microarray data indicates that the expressions level of Bmagxt2in each tissue of fifth-instar day3larvae are very low, besides a gleam expression in ovary, testis and fat body.The gene of Bmhyi has different expression level in silkgland, ovary, testis, malpighian tubules, body wall, head, blood, fat body and midgut. Among them, the expression level in silkgland is low, but is high in malpighian tubules, blood, fat body and midgut, especially in malpighian tubules. The gene of Bmhibadh has hardly expressed in these tissues of day-3in fifth-instar of larvae. The expression data from early embryo, the mature stage to the moths stage indicates that there exists no datum in the developmental stage and early embryo. The expressions level of Bmagxt2is low in early embryo and on the day-8to9of mounting stage but hardly appear in other stages. In contrast, the gene of Bmhyi has no expression in the whole embryonic period.For the purpose of the further addition and verification of the expression profiling, we made use of Semi-quantitative RT-PCR before. Bmagxt2is widely expressed in gonad, body walland fat body in fifth-instar day3larvae, while the expression level of the head and malpighian tubules is low, though there has expression from1st molting stage to day-75th stage of larve. The expression level of Bmhyi is low in fat body, epidermis and blood cell, but, On the contrary, the data of malpighian tubules is high. There has the expression during the period of ant silkworm and the day-10of mounting stage. The whole process almost shows that the expression level of larval stage is higher than that of pupal stage, but the level after36-72h of mounting stageis lower than that after96h to the day-10of mounting stage. Bmhibadh is expressed in all the tissues during fifth-instar day3larvae, but the expression level in the malpighian tubules and silkgland is a little higher than that in other tissues. However, the larval stage expresses a certain amount of data, and the overall trend is that the expression level of sleep period is lower than that of wandering stage. It hardly has no expression after on mounting stage, until the day-8of mounting stage to moths stages.5. Prokaryotic expression and antibody preparation of Bmagxt2, Bmhyi and wester blotting examinationFor the further research regarding gene function, the cDNA of Bmagxt2and Bmhyi were respectively connected with p29and p28to construct expression vector, which named as Bmagxt2/p29and Bmhyi/p28, and transformed into expression strain BL21(DE3). After induced by IPTQa51kD and a30kD recombinant protein were obtained. However, the target protein molecular weights are about50.38kD and29.17kD. In addition of a6×His-tag and a MBP-tag, the molecular weights are about51kD and30kD, which is consistent with the forecasted molecular weights.We obtained high concentration Bmagxt2and Bmhyi with the methods of affinity chromatograph and electroelution. With these two proteins, immunize male rabbits, get the serum, and purify polyclonal antibodies. Then, examin the expression level of the Bmagxt2and Bmhyi in tissues with the antibodies on day-5of larve. The research comes out that Bmagxt2protein has expression in blood and especially high in malpighian tubules, which is consistent with the phenomenon in the experiment of two-dimensional electrophoresis. In contrast, Bmhyi protein only expressed in malpighian tubules. However, the further western blotting results shows that, compared with the condition on day-5of larve, Bmagxt2and Bmhyi in malpighian tubules reduced in a huge degree on the first day in pupal stage, which is consistent with the results of two-dimensional electrophoresis.6. Detecting the activity of alanine-glyoxylate aminotransferase2(BmAGXT2)In order to study BmAGXT2protein activity in the malpighian tubules its expression of the correlation, we refer to the homologous proteins in human and mouse activity was measured, and measured the activity of BmAGXT2in malpighian tublues from day-3fifth instars larvae to day-7pupa and in each section on day-5fifth instars larvae. The results indicate that:1.The activity of BmAGXT2increased from day-3to day-7fifth instars larvae, then reduced throughout the pupal stage and stable in a relatively low level.2. In larval malpighian tublues, the activity of BmAGXT2the highest in the first section, and the lowest in the third sectin. Suggesting that BmAGXT2may be associated with metabolic intensity and silkworms eat mulberry leaves have a certain degree of correlation. BmAGXT2play its role mainly in the first section of the the silkworm, suggesting that BmAGXT2may be play its role mainly in the first section of malpighian tublues..更多还原