【作者】 刘利；

【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】 PAT家族蛋白是脂肪滴(lipid droplet,LD)表面主要结构蛋白,在脂肪代谢过程中具有重要的作用,参与脂滴的合成、分解和转运过程。目前,关于PAT家族蛋白的研究主要集中在哺乳动物中,而在昆虫中的报道很少。在哺乳动物中发现的PAT家族蛋白成员包括脂滴包被蛋白(perilipin)、脂肪分化相关蛋白(ADRP)、47kD的尾连蛋白(TIP47)、S3-12和氧化型PAT蛋白(OXPAT)。昆虫的基因组只能编码两种PAT家族蛋白,即脂肪储存小滴蛋白1(LSD-1)和脂肪储存小滴蛋白2(LSD-2)。LSD-1作为昆虫脂滴表面重要的PAT家族蛋白直接参与脂肪代谢的调控。本文以中华蜜蜂(Apis cerana cerana)为实验材料,首次从中华蜜蜂中克隆得到LSD-1基因(AccLSD-1),并对其进行了序列比对,表达特性分析及初步功能鉴定,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、利用RT-PCR和RACE-PCR的方法,首次从中华蜜蜂中克隆得到了LSD-1基因,命名为AccLSD-1。并将该基因在Genbank注册,其注册号为GU722328。序列分析结果表明,AccLSD-1cDNA全长为1313 bp,包括1161 bp的开放阅读框(ORF),40 bp的5′非编码区(5′UTR)和112 bp的3′非编码区(3′UTR)。该基因编码一个386个氨基酸残基的多肽,预测分子量为42.6 kDa。通过同源性比较发现,中华蜜蜂LSD-1与其它昆虫LSD-1的同源性很高。AccLSD-1含有昆虫LSD-1s家族所有的PKA磷酸化位点和脂肪滴结合位点。进化分析表明,AccLSD-1与昆虫LSD-1聚集成簇,而与哺乳动物PAT家族蛋白的进化关系较远。AccLSD-1基因组全长5224 bp,包含8个外显子和7个内含子。其中,内含子1位于5′UTR内,推测该内含子可能具有特殊的功能。2、通过LA-PCR和I-PCR的方法获得AccLSD-1基因的启动子序列。利用分析软件TFBIND对其进行分析,发现该序列除了存在典型的TATA box和CAAT box外,还存在多个响应脂肪代谢的特殊作用元件,如PPARγ响应元件和C/EBPα元件。另外,还发现了几个响应环境应激的作用元件,如热响应元件和T细胞响应元件。3、利用Real-time PCR的方法,研究了AccLSD-1在不同发育时期mRNA水平的表达情况。研究表明,AccLSD-1在中华蜜蜂的整个发育过程中均有表达。在幼虫时期(3日龄至5日龄),AccLSD-1表达量随日龄的增加逐渐升高。在成虫时期,AccLSD-1在25日龄青年蜂的表达量明显高于2日龄幼蜂和50日龄老年蜂的表达量。然而,在蛹期AccLSD-1表达呈现抛物线的趋势且在Pb时期有最高表达量。同时还发现在中华蜜蜂蜕变时期(无论是幼虫蜕变成蛹还是蛹蜕变成成虫)AccLSD-1的表达量都明显降低。4、利用室内人工饲养的方法,检测了AccLSD-1在不同浓度的共轭亚油酸或罗格列酮诱导下mRNA水平的表达情况。结果表明,AccLSD-1转录水平受罗格列酮的诱导而升高,而添加共轭亚油酸能降低AccLSD-1的表达量。与只添加共轭亚油酸组相比,同时添加共轭亚油酸和罗格列酮能明显提高AccLSD-1的表达量。5、从AccLSD-1中选取N端600氨基酸片段构建原核表达载体pET- AccLSD-1-600,并在大肠杆菌BL21(DE3)中表达融合蛋白,将强诱导带切下,溶于PBS中获得抗原,并免疫小鼠制备抗体,其抗血清效价为1:2000。本研究分析了AccLSD-1基因的结构特征,并进行了功能预测。根据试验结果,我们推测AccLSD-1在中华蜜蜂蜕变发育,尤其在蛹的发育过程中发挥重要作用,且共轭亚油酸或罗格列酮可能通过PPARγ途径调控AccLSD-1表达。更多还原

【Abstract】 PAT family proteins, the major structural proteins located in the coat of lipid droplet (LD), play an important role in lipid metabolism and are involved in lipid droplets formation, decomposition and transport. Recent studies on PAT family proteins mainly foucs on the mammal, yet little is known about these in insects. In mammal, five PAT family proteins including perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT have been identified by a number of previous studies. However, only two PAT proteins, lipid storage droplet protein-1 and -2 (LSD-1 and LSD-2), are expressed by insect genomes. LSD-1, a PAT family protein located around LD in insects, is linked to control of lipolysis. In this study, we select the Apis cerana cerana as the experiment material, and a series of research have been managed on the isolation, sequence and expression profile analysis, and functional identification of AccLSD-1, which can greatly help to study the function and mechanism of LSD-1. The main results are as follows:1. Through RT-PCR and RACE-PCR methods, a novel LSD-1 gene, named as AccLSD-1 (GenBank accession no: GU722328), was isolated from Apis cerana cerana. The result of sequence analysis indicated that the full-length cDNA was 1,313 bp in size and contained an open reading frame (ORF) of 1,161 bp. There was a 40 bp 5′untranslated region (UTR) upstream of the start codon and a 112 bp 3′UTR following the stop codon. The deduced AccLSD-1 protein was 386 aa along with a calculated molecular weight (MW) of 42.6 kDa. Multiple sequence alignment showed that AccLSD-1 had high homology with other insect LSD-1s, and AccLSD-1 also contained lipid binding spot and PKA-phosphorylation sites. Phylogenetic analysis revealed that AccLSD-1 had closer relation with insect LSD-1 than other mammal PAT family proteins. The genomic DNA of AccLSD-1 was 5,224 bp in length, including 8 exons and 7 introns. Interestingly, the first intron of AccLSD-1 was located in the 5′UTR, which suggested that the first intron might play an important role in transcription of AccLSD-1.The promoter sequence of the AccLSD-1 gene was obtained by LA PCR and reverse PCR. Using the analysis soft TFBIND, we find that in addition to the typical TATA-box and CAAT-box, the binding sites of PPARγand CAAT/enhancer binding proteinα(C/EBPα), which are the major and determining adipogenic transcription factors, were found in this region. In addition, several important transcription factors such as heat shock factors (HSF) and T-cell factor required for regulating various environmental stresses were predicted.2. The expression profile of AccLSD-1 at the different developmental stages was detected by real-time quantitative PCR. The results indicated that AccLSD-1 constitutively expressed throughout feeding larval, pupal and adult stages. In the larval feeding stages (L3-L5), the AccLSD-1 transcripts showed a very steep growth as increasing ages of larvae. In adult stage, the expression of AccLSD-1 exhibited much stronger in youth workers (25 day-old) than childhood (2 day-old) and old age ones (50 day-old). Howerver, AccLSD-1 was transcribed in a parabola fashion rather than persistently reduced trend in nonfeeding pupal stage and exhibt a highest level in brown-eyed pupae (Pb) stage. Moreover, there was an abrupt decrease in the quantity of transcripts in both larval-pupal transition and pupal-adult transition.4. The effects of various concentrations of conjugated linoleic acid (CLA) or rosiglitazone (Rosi) diets on the expression pattern of AccLSD-1 were investigated through rearing honeybee in laboratory. The results revealed that the transcripts of AccLSD-1 could be up-regulated by Rosi and down-regulated by CLA. The congenerous application of CLA and Rosi showed obvious alteration of the AccLSD-1 expression that was significantly higher than that of CLA alone group.5. A 600 aa 5′fragment of AccLSD-1 was selected to construct an E.coli expression vector pET- AccLSD-1-600, and then it was induced by IPTG to express in E.coli strain BL21 (DE3). The strong induced fusion protein bands were collected into PBS solution and immuned smart mouse to obtain antiserum. The value of antibody reaches 1:2000.This study identified the structural characterization and functional implication of a novel AccLSD-1 gene, showing that AccLSD-1 plays a considerable role in A. cerana cerana development, especially during pupal metamorphosis, and can be regulated by CLA or Rosi possibly via activating peroxisome proliferator–activated receptor-γ(PPARγ).更多还原