草鱼脑芳香化酶的cDNA克隆、启动子分离与转录活性的调控
摘要
细胞色素P450芳香化酶能够催化雄性激素向雌性激素的转化,被认为是类固醇激素代谢过程中的一个关键酶。在硬骨鱼中存在两种芳香化酶异构体,P450aromA和P450aromB,分别在性腺组织和脑组织中有高表达。有趣的是,硬骨鱼的脑芳香化酶活性大约是其他脊椎动物的100-1000倍,但至今对这种现象未有合理的解释。在目前的研究中,我们运用RT-PCR以及快速扩增cDNA末端(RACE)的方法成功克隆了草鱼脑芳香化酶(gcP450aromB)的cDNA序列。该序列全长共有1947bp,其中开放阅读框(ORF)长1527bp,可以编码509个氨基酸残基。经氨基酸序列比对发现,草鱼脑芳香化酶与其他硬骨鱼类脑芳香化酶有较高的相似性(66%-93%),而与性腺芳香化酶仅有约60%相似性,与哺乳动物芳香化酶有50%的相似性。运用半定量RT-PCR的方法进行组织分布研究发现,在所选择的10个组织中,仅在脑和垂体腺中发现了gcP450aromaB的mRNA表达,进一步在脑的不同区域中发现gcP450aromB的mRNA在视前区、下丘脑和垂体区域有表达。此外,利用基于PCR方法的基因步移法克隆了草鱼脑芳香化酶基因5’端的上游序列。所获得的启动子序列共有2.3kb,其中包括了1个TATA框,1个CAAT框以及许多潜在的转录因子结合位点,包括GATAs,AP1,ERE, CRE,C/EBP,NF-1,Sp1,PPARalpha/RXR-alpha,SREBP-1,GP,NF-kappaB,AhR/Arnt,STATx元件和CRE-BP1/c-Jun等等。此外,实验结果显示,gcP450aromB基因的翻译从第二个外显子开始,与不翻译的第一个外显子之间存在一个较长的内含子,大约有2.7kb。草鱼脑芳香化酶启动子活性的检测是以体外大鼠神经胶质瘤细胞(C6)为模型,对5’端上游序列采取5’端逐渐缺失片段构建荧光素酶报告基因的表达质粒来完成。结果显示,与阴性对照pGL-3-basic相比,几乎所有包括了第一个内含子全部序列的报告质粒都表现出不同程度的转录活性,而仅含有启动子序列的的报告质粒都没有转录活性。实验中还初步探讨了下丘脑分泌因子(PACAP)和垂体分泌激素(STH、GTH和activin)对调控序列转录活性的影响,表明PACAP在调控序列缺失到pGL3-159/+2822后一直表现为明显的抑制作用,而GTH对不同长短的启动子序列的转录水平都呈现出抑制作用,特别是在pGL3+1855/+2822抑制效果尤为显著。与之不同的是,生长激素(STH)在报告质粒pGL3-828/+2822显示出明显的促进作用。但是这种作用在进一步的缺失后被阻断,甚至在+861/+1855区域缺失后转为抑制作用。出乎意料的是,activin对草鱼脑芳香化酶的转录调节效果与生长激素的相类似。
As cytochrome P450 aromatase can catalyze the conversion of androgens to estrogens, it is considered as a key enzyme in the hormonal steroidogenic pathway. In teleosts, two isoforms of aromatase are expressed mainly in ovary and brain, which are named as aromA and aromB respectively. Interestingly, teleost fish aromB expression is 100-1000 times greater than that found in other vertebrates, but the mechanism responsible for this phenomenon is unclear. In the present study, the full-length cDNA of aromB was cloned from grass carp by RT-PCR coupled to rapid amplification of cDNA ends, named as gcAromB. The cDNA contained an open reading frame of 1527bp encoding a predicted protein of 509 amino acid residues, which shared 66-93% sequence identity with its counterparts in other teleosts, but only showed about 50% identity to mammalian aromatase. RT-PCR analysis revealed that gcAromB only expressed in brain from 10 selected tissues. Subsequently, aromB mRNA was detected in the partial areas of grass carp brain, including the optic tectum, hypothalamus and pituitary. Furthermore, A PCR-based genomic walking strategy was used to isolate the 5’-flanking region of grass carp aromB. The grass carp aromB 5’flanking region (2.3 kb) contained a consensus TATA box, a CCAAT box and some potential transcription factor binding sites including some GATAs, four AP1, an ERE, two CRE, four C/EBP, two NF-1, two Sp1, a PPARalpha/RXR-alpha, a SREBP-1, a GP, a NF-kappaB, an AhR/Arnt, three CRE-BP1/c-Jun and three STATx elements. The transcription activity of gcAromB was evaluated in vitro using luciferase as the reporter gene. In the glioma cells (C6) transfected by constructs with decreasing length of gcAromB promoter (-2290/+164), unexpectively, the basal levels of luciferase activity could not be detected. In contrast, all reporter constructs with intron I transiently transfected into glioma C6 showed differentical basal activity. To test effects of hypothalamus factor and pituitary hormones on the transcription activity of gcAromB, C6 cells transfected by constructs were exposed to PACAP, GTH, GH and activin. In this case, both PACAP and GTH significantly reduced the promoter activity from pGL3-159/+2822 to pGL3+1855/+2822, and this effect was blocked by the following deletion, and then reversed due to the absence of +861/+1855 region. Consistently, activin exhibited similar effects on promoter activity of gcAromB as GH.
As cytochrome P450 aromatase can catalyze the conversion of androgens to estrogens, it is considered as a key enzyme in the hormonal steroidogenic pathway. In teleosts, two isoforms of aromatase are expressed mainly in ovary and brain, which are named as aromA and aromB respectively. Interestingly, teleost fish aromB expression is 100-1000 times greater than that found in other vertebrates, but the mechanism responsible for this phenomenon is unclear. In the present study, the full-length cDNA of aromB was cloned from grass carp by RT-PCR coupled to rapid amplification of cDNA ends, named as gcAromB. The cDNA contained an open reading frame of 1527bp encoding a predicted protein of 509 amino acid residues, which shared 66-93% sequence identity with its counterparts in other teleosts, but only showed about 50% identity to mammalian aromatase. RT-PCR analysis revealed that gcAromB only expressed in brain from 10 selected tissues. Subsequently, aromB mRNA was detected in the partial areas of grass carp brain, including the optic tectum, hypothalamus and pituitary. Furthermore, A PCR-based genomic walking strategy was used to isolate the 5’-flanking region of grass carp aromB. The grass carp aromB 5’flanking region (2.3 kb) contained a consensus TATA box, a CCAAT box and some potential transcription factor binding sites including some GATAs, four AP1, an ERE, two CRE, four C/EBP, two NF-1, two Sp1, a PPARalpha/RXR-alpha, a SREBP-1, a GP, a NF-kappaB, an AhR/Arnt, three CRE-BP1/c-Jun and three STATx elements. The transcription activity of gcAromB was evaluated in vitro using luciferase as the reporter gene. In the glioma cells (C6) transfected by constructs with decreasing length of gcAromB promoter (-2290/+164), unexpectively, the basal levels of luciferase activity could not be detected. In contrast, all reporter constructs with intron I transiently transfected into glioma C6 showed differentical basal activity. To test effects of hypothalamus factor and pituitary hormones on the transcription activity of gcAromB, C6 cells transfected by constructs were exposed to PACAP, GTH, GH and activin. In this case, both PACAP and GTH significantly reduced the promoter activity from pGL3-159/+2822 to pGL3+1855/+2822, and this effect was blocked by the following deletion, and then reversed due to the absence of +861/+1855 region. Consistently, activin exhibited similar effects on promoter activity of gcAromB as GH.
引文
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