山羊clock和cry1基因的克隆及其在大脑和垂体中表达差异的研究

Molecular Cloning and Differential Expression of clock and cry1 Genes in Goat Brain and Pituitary

本研究旨在探讨生物钟基因clock和cry1在山羊出生后不同时期的大脑和垂体中的表达差异。在出生后30、60、90和120d共屠宰24只南江黄羊羔羊(公母各12只),取其大脑皮质层和垂体组织样品用于抽提RNA,进行分子克隆和实时荧光定量PCR分析。结果,分离克隆得到了山羊clock基因外显子1到外显子8以及cry1基因外显子6到外显子15的CDS区序列,其长度分别为1 479bp和993bp,其中clock基因部分CDS区序列与绵羊、牛、人和小鼠的对应序列相似性分别为99%、90%、94%和92%,cry1基因部分CDS区序列与绵羊、牛、人和小鼠的对应序列相似性分别为99%、98%、94%和87%。组织表达分析结果表明,性别对clock和cry1基因的表达影响不显著(P>0.05),2个基因在垂体中的mRNA表达量极显著高于大脑中的表达量(P<0.01);在不同时期的垂体组织中,clock和cry1基因表达量均呈现先上升后下降的趋势;在不同时期的大脑组织中,clock基因的表达量趋于波动平衡,呈现上升趋势,cry1基因的相对表达量呈细微的上升趋势。结果提示:clock和cry1基因的CDS区在物种间保守性较高。clock和cry1基因在山羊大脑和垂体组织中起到重要的作用,且其mRNA发育性表达模式具有组织特异性。

The objective of the present study was to investigate the differential expression of clock and cry1 genes in the brain and pituitary of postnatal goats. The samples of cerebral cortex and pituitary were collected from a total of 24 （12 males and 12 females） Nanjiang Yellow goats at 30,60,90 and 120 days of age after birth. The mRNA levels of clock and cry1 genes were detected by real-time fluorescence quantitative PCR. The partial CDS of clock and cry1 genes were obtained by RT-PCR, containing exon 1 to exon 8 in clock gene and exon 6 to exon 15 in cry1 gene, and the lengths were 1 479 and 993 bp,respectively. The nucleotide sequence of CDS shared 99%, 90%, 94% and 92% consistency with the clock gene of sheep, cattle, human and mouse, respectively. Whereas the nucleotide sequence of CDS shared 99%, 98%, 94% and 87% consistency with the cry1 gene of sheep, cattle, human and mouse. The mRNA levels of clock and cry1 genes in pituitary were significantly higher than that in brain（P〈0.01）, however there was no significant difference（P〉0.05） between sexes. In pituitary, the mRNA levels of clock and cry1 genes were upregulated from 30 to 60 days, and then decreased. In addition, the expression of clock gene showed no significant difference at four development stages of brain, but the cry1 gene was upregulated slightly from 30 to 120 days. These results indicate that clock and cry1 genes are highly conservative, and play important roles in the brain and pituitary in goats. The mRNAs express developmentally in a tissue-specific manner.