猪ALAS1基因的克隆、序列分析及表达模式的研究  

Cloning,Sequence Analysis and Expression Pattern of Porcine ALAS1 Gene

作　　者：刘林清 李凤娥[2] 熊远著[2] 邓昌彦[2] 

机构地区：[1]安徽省农业科院畜牧兽医研究所,安徽合肥230031 [2]华中农业大学动物科学院,湖北武汉430070

出　　处：《养猪》2015年第4期69-72,共4页Swine Production

基　　金：院长青年创新基金面上项目(14B0427);安徽省农业科学院科技创新团队项目(13C0405);现代农业子项目"安徽省地方品种猪保护与利用"资金资助(201201)

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9137 bp，其中包括53 bp的5＇非编码区序列、1922 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。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 10 PKC_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.

关 键 词：猪 ALAS1 序列分析 表达模式 

分 类 号：S828[农业科学—畜牧学]