摘要
文昌鱼被认为是现存与脊椎动物最接近的原索动物,代表着从无脊椎动物到脊椎动物进化过程中的一个重要的过渡阶段,是研究脊椎动物起源与进化的经典实验模型。利用分子生物学手段,研究文昌鱼有关基因的结构、进化和表达,可为我们从分子水平上解开脊椎动物起源的奥秘。更重要的是,将进化与发育联系起来,可以更清楚地揭示有关基因在发育中的作用。
铁蛋白Ferritin的主要生理功能是储存机体中的过剩铁,避免产生铁中毒和释放铁给需铁的细胞,用于体内生物合成含铁的蛋白质或酶,同时还可以防止铁双氧化过程中所产生的自由基带来的伤害。编码这一蛋白的基因已从人、动物、植物、真菌和细菌中得到分离。迄今为止,原索动物的铁蛋白却未见报道。
我们在获得文昌鱼铁蛋白基因序列的基础上,通过切片原位杂交和免疫组织化学方法研究了该基因在文昌鱼体内的表达,进而对文昌鱼铁蛋白基因的功能和系统进化进行研究。
文昌鱼铁蛋白(BbFRT)cDNA序列全长1038 bp,含有一个519 bp的开放阅读框(ORF),编码一个172氨基酸残基组成的多肽,分子量约为20.0kDa。在cDNA 5’端非翻译区有116个碱基, 3'端非翻译区为403个碱基,有一个终止子,并含有一个多腺苷酸信号AATAAA和一个poly(A)尾。起始密码子ATG位于118～120位核苷酸,并且在-3和+4位置均为嘌呤,符合典型的kozak结构。这就表明该cDNA编码一个全长蛋白质。对BbFRT cDNA 5’端非翻译区进行分析,发现在起始密码子ATG上游73 bp处存在一个铁蛋白特有的铁反应元件(iron responsive element,IRE)。该元件含有保守的CAGUGN环状结构和一个CYS,其mRNA二级结构形成典型的柄环结构。对NCBI数据库进行BLASTp相似性搜索发现该蛋白氨基酸序列与脊椎动物铁蛋白H亚基氨基酸序列同源性为68%,与L亚基氨基酸序列同源性为46-51%,与一些已报道的无脊椎动物铁蛋白氨基酸序列同源性为72-77%。
系统进化分析表明,无脊椎动物和脊椎动物的铁蛋白各自聚为一个类群,而BbFRT蛋白与无脊椎动物铁蛋白聚为一类,说明其与无脊椎动物铁蛋白在进化地位上更加接近。
将文昌鱼BbFRT基因插入pET28a融合表达载体,在原核E. coli中实现了高效稳定的表达,并纯化出BbFRT融合蛋白。利用该融合蛋白制备的多克隆抗体与文昌鱼的体液中的总蛋白进行Westen blot杂交,出现较强信号,说明在文昌鱼体内含有BbFRT蛋白,而且该抗体具有独特的专一抗性。
切片原位杂交和免疫组织化学实验都显示BbFRT在文昌鱼体内广泛表达。
对BbFRT的功能进行分析,发现细菌内毒素LPS处理文昌鱼后,BbFRT基因的表达量在转录水平和翻译水平上相对于对照分别升高了1.5倍和1.6倍。铁处理文昌鱼后,BbFRT基因的表达量在翻译水平上相对于对照升高了1.6倍。表明BbFRT基因表达能够被LPS和铁诱导。这就说明BbFRT不仅能够调节文昌鱼体内铁的代谢,而且对LPS起急性应急反应,在体内起免疫作用。
Amphioxus or lancelet, a protochordate, has long been regarded as a model animal for gaining understanding of the origin of vertebrates. Its genetic information on gene sequence and expression pattern has been widely used for interspecies comparative genome studies and developmental homology analysis.
Ferritin plays a key role in cellular iron metabolism including iron storage and detoxification, which has been identified in a wide range of organisms including bacteria, fungi, plants and animals. However, little information is available regarding ferritin in the protochordates to date.
The cDNA (GenBank accession number: AAQ21039) obtained from the gut cDNA library of B. belcheri is 1038 bp long, and its longest open reading frame (ORF) was 519 bp, corresponding to a deduced protein of 172 amino acids with a predicted molecular mass of approximately 20 kDa. The 5’-untranslated region (UTR) was 116 bp long, and the 3’-UTR 403 bp long with a polyadenylation signal AATAAA and a polyadenylation tail . The initiation codon (ATG) was assigned on the basis that there is no ATG in the 5’-UTR 116 nucleotides and the DNA surrounding the initiation codon AT G has a purine at positions of both -3 and +4, which is in accordance with the Kozak consensus sequence. Therefore, cDNA encodes a full-length sequence protein. Analysis of the 5’-UTR demonstrated the presence of a putative iron-responsive element (IRE) in the 5'UTR of BbFRT 73 bp upstream of the start codon ATG, which includes a typical CAGUGN loop and a bulged Cys. The predicted secondary structure of BbFRT IRE mRNA was a typical stem loop structure like that identified in most ferritin mRNAs.
Sequence comparisons showed that BbFRT was more identical to H-chains (68%) of vertebrate ferritins than to the L-chains (46-51%). It also shared 72-77% identity to invertebrate ferritins documented so far. Phylogenetic analysis revealed two separate clusters of vertebrate and invertebrate ferritins . The new ferritin BbFRT was grouped together with the invertebrate ferritin cluster.
An expression vector including the entire open reading frame of BbFRT and a 5’additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. Rabbit antisera against the purified BbFRT were obtained. Western blotting analysis exhibited that the rabbit antisera reacted with B. belcheri humoral fluids, producing a ~20 kDa band, corresponding to the molecular mass predicted by BbFRT cDNA. These denoted that the rabbit antisera prepared display a conspicuous antigen-specific reactivity.
Both in situ hybridization histochemistry and immunohistochemical staining revealed that BbFRT was ubiquitously expressed in B. belcheri.
In addition, BbFRT expression was up-regulated by 1.6-fold and 1.5-fold, respectively, following exposure to LPS and iron at both transcriptional and translational levels. These suggest that BbFRT seems a protein with a dual function functioning in both immune response and iron metabolism.
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