【摘要】 目的:首先克隆了猪ALAS1(5-aminolevulinate synthase 1)DNA序列,并对CDS序列进行了序列分析,分析了该基因的表达模式。方法:以4月龄长大母猪的卵巢为材料,运用同源序列克隆技术,对猪ALAS1 DNA序列进行克隆并对其进行生物信息学分析;利用性腺激素处理卵巢颗粒细胞并结合RT-PCR方法分析了猪ALAS1基因的表达模式。结果:克隆得到了猪ALAS1 DNA序列9 137 bp,其中包括53 bp的5’非编码区序列、1 922 bp的CDS序列和2~9内含子序列,共编码640个氨基酸;猪ALAS1蛋白氨基酸序列与人和小鼠的相似性一致(100%);ALAS1蛋白的分子量约为38.0 KDa,等电点为9.07,可能存在10个PKC磷酸化位点、1个CAMP磷酸化位点、4个N-糖基化位点、12个豆蔻酰化位点、3个CKII磷酸化位点和1个酰胺位点,还包含1个天冬酰胺转氨酶超家族(AAT-1)保守结构域;猪ALAS1在性腺激素处理2个卵巢颗粒细胞后,可检测到该基因在卵巢颗粒细胞中的显著性表达,之后其表达量迅速下降,12 h后表达量又提高,之后表达量再次迅速下降。结论:猪ALAS1基因的CDS区在物种间保守性强,推测猪ALAS1基因参与卵泡的发育和排卵及在黄体化过程中发挥一定作用。更多还原

【Abstract】 Objective: The genomic sequence of porcine ALAS1 gene was cloned and temporal and spatial expression pattern of ALAS1 gene was studied.Method:According to the related CDS sequences of other species were measured.The structure of the porcine ALAS1 gene analyzed by bioinformatics methods.The expression pattern of ALAS1 gene were analysis by RT-PCR by treatment with PMSG/HCG in ovarian granulosa cells.Result:We cloned a 9 137 bp genomic sequence of ALAS1 gene,which containing 53 bp of 5’ untranslated sequence,its 2 ~9 intron sequences,1 922 bp complete open reading fragment(ORF) encoding a protein of 640 amino acids.The amino acid sequences exhibited the greatest identity with Human(100%) and mus(100%).The molecular weight,PI of ALAS1 were 38.0 KDa and the predicted isoelectric point is 9.07.There were 10PKC_PHOSPHO_SITE,1 CAMP_PHOSPHO_SITE,4 N-glycosylation site,12 N-myristoylation site,3 CK2_PHOSPHO_SITE,1 amidation site and contain one conserved domains(AAT-1) in ALAS1 protein.The highly levels of ALAS1 gene are observed at 2 h,12 h by PMSG/HCG,and the time of follicular development and the low level ALAS1 gene are observed at 4 ~10 h and 16 ~24 h by PMSG/HCG,and the time of ovulation and follicle luteinization.Conclusion:The result indicates that ALAS1 gene is highly conservative among species and the porcine ALAS1 involved in the regulation of follicular development,ovulation and even luteinizing process,plays a role in the process of ovulation.更多还原