大口黑鲈GHRH基因启动子区域序列分析及其活性检测

Sequence and activity analysis of GHRH promoter region from Micropterus salmoides

生长激素释放激素(growth hormone releasing hormone,GHRH)是下丘脑弓状核合成和分泌的小分子多肽,其主要功能是调节垂体细胞合成和释放生长激素。为研究大口黑鲈(Micropterus salmoides)GHRH基因5’侧翼启动子区域的活性和该区域中潜在的转录因子对GHRH基因表达的调控作用,对该基因5’端启动子区域约1400 bp长度的片段进行序列分析,预测顺式作用元件,获得了Oct-1、SP1、NF-1、C/EBPalp和C/EBP等多个潜在的调控GHRH基因表达的调节因子结合位点序列。在包括外显子1和内含子1的GHRH基因5’侧翼区两侧加入两个限制性酶切位点Xho I和Bam H I,对其进行改造,并将该片段插入红色荧光蛋白报告基因载体p DsRed2-1,构建了重组表达质粒pGHRH1-RFP。同时,用不含有外显子1和内含子1的GHRH基因5’侧翼区构建重组表达质粒p GHRH-RFP。将质粒pGHRH1-RFP和p GHRH-RFP转染鲤(Cyprinus carpio)上皮细胞(epithelioma papillosum cyprinid,EPC)。经过48 h的培养,在pGHRH1-RFP转染的部分细胞中检测到红色荧光蛋白表达。又将pGHRH1-RFP或p GHRH-RFP质粒注射到斑马鱼(Danio rerio)一细胞或二细胞期的胚胎中,注射了pGHRH1-RFP的胚胎在受精后48 h约有22.5%能检测到有红色荧光蛋白表达,受精后72 h约有29%的仔鱼检测到红色荧光蛋白表达。实验结果表明,目前分离到的GHRH基因5’侧翼序列具有启动基因表达的活性,且该基因的内含子1和外显子1是启动子的活性所必需的。另外,pGHRH1-RFP质粒注射的斑马鱼胚胎只能在胚胎和仔鱼的脊椎和肌肉中检测到RFP的表达,而在脑中没有检测到表达。推测扩增到的大口黑鲈GHRH启动子序列1407 bp(-1043 bp^362 bp)只是起到了驱动RFP脊椎和骨骼肌表达的作用,而不包括驱动在脑组织中特异性表达的启动子,本研究为GHRH基因功能的深入分析奠定了基础。

Growth hormone releasing hormone （GHRH）is a small molecular weight peptide released from the arcuate nucleus of hypothalamus. It mainly plays the role to regulate the synthesis and release of growth hormone （GH） from the anterior pituitary somatotrophs. Non-pituitary GHRH has a wide spectrum of activity, including modulating cell proliferation, especially in malignant tissues, regulating differentiation of some cell types, and promoting healing of skin wounds. In order to analyze the activity of GHRH 5＇ flanking region and the mechanism of the potential transcription factors regulating GHRH gene expression in largemouth bass （Micropterus salmoides）, the cis-acting elements were predicted by the transcription element search system for the 1400 bp-length fragment in 5＇ flanking region. Many sites related to regulating the GHRH expression were identified, including multiple of Oct-1, SP1, NF-1, C/EBPalp and C/EBP binding sites, which indicated that these sites probably played the key roles to regulate the spatiotemporal expression of GHRH. The GHRH 5＇ flanking region fragment with integrate exon 1 and intron 1 was modified by adding to two restrict enzyme sites, Xho I and BamH I, and was inserted into the red fluorescent protein （RFP） reporter gene vector pDsRed2-1. Then the recombined plasmid pGHRH1-RFP was constructed. Meanwhile, the recombinant plasmid pGHRH-RFP was constructed using the fragment without exon 1 and intron 1. The plasmids pGHRH1-RFP and pGHRH-RFP were transfected epithelioma papillosum cyprini （EPC） cells. After 48 h of culture, the expression of the RFP was detected in EPC cells with pGHRH1-RFP, but not in the cells as negative controls or the cells transfected by pGHRH-RFP. At the same time, the plasmid pGHRH1-RFP or pGHRH-RFP was also microinjected in zebrafish embryos at the one cell or two cell stages. Embryos without microinjection served as negative controls. A total of 22.5% （n =45/200） embryos injected with pGHRH1- RFP could be detected RFP 48hpf （hours post fertilization） and 29% （n = 16/56） fries could be detected RFP 72hpf. And the RFP was not detected in the zebrafish embryos and larvae microinjected with plasmid pGHRH-RFP and negative controls. The results showed that the isolated GHRH 5 ＇ flanking region had the activity of starting GHRH gene expression. And there are indispensable cis-elements in exon 1 and intron 1 of GHRH gene for promoter activity. Additionally, it is interesting that the RFP is found in spine and muscle, not in brain. In rat, GHRH gene is spliced into alternative upstream promoters in brain, gonads and placenta. It suggests that the cis-acting elements responsible for GHRH spatially specific expression in brain of largemouth bass have not been discovered yet. The current study can lay foundations for analysis of the functions of GHRH gene.