目的通过研究丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因mRNA及其编码蛋白产物在小鼠睾丸组织中的表达特征,探讨该基因在精子发生过程中的作用及意义。方法分别采用半定量逆转录聚合酶链反...目的通过研究丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因mRNA及其编码蛋白产物在小鼠睾丸组织中的表达特征,探讨该基因在精子发生过程中的作用及意义。方法分别采用半定量逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹杂交(Western blotting)分析该基因mRNA及蛋白产物在小鼠多种组织中的表达;利用实时定量PCR(real-time quantitative PCR)分析SRPK2 mRNA在不同发育阶段小鼠睾丸组织中的差异表达;应用免疫组织化学染色和间接免疫荧光技术观察SRPK2蛋白在小鼠曲精小管中的细胞定位和生精细胞内的亚细胞定位。结果半定量RT-PCR和Western blotting分析显示SRPK2 mRNA和蛋白在小鼠睾丸组织中均大量表达;实时定量PCR分析发现SRPK2 mRNA在5周及8周龄雄性小鼠睾丸组织中显著表达,具有明显的阶段特异性表达特征。免疫组织化学染色结果表明SRPK2蛋白阳性着色主要位于曲精小管中的长形精子细胞核;间接免疫荧光分析显示SRPK2蛋白定位于长形精子细胞核表面。结论 SRPK2基因在小鼠睾丸组织中大量表达,并且具有显著的阶段特异性表达特征和明确的细胞核定位,极有可能在小鼠精子发生的变态成形期参与mRNA前体分子的剪接过程,其作用机制值得进一步深入研究。展开更多
Part 5＇ UTR region of pig SRPK1 gene was cloned by inverse PCR （I-PCR）, then a 425 bp gene sequence was acquired. A promoter region in -1--309 bp was predicted by online tool （TFSEARCH） and 32 binding sites of tr...Part 5＇ UTR region of pig SRPK1 gene was cloned by inverse PCR （I-PCR）, then a 425 bp gene sequence was acquired. A promoter region in -1--309 bp was predicted by online tool （TFSEARCH） and 32 binding sites of transcription with scores higher than 85 were getten, of which scores of Spl, MyoD, and HSF2 were over 90. Some of these binding sites of transcription factors were connected with promoters, but TATA-box, which was important to gene expression, hadn＇t been found in this region. By using PCR-SSCP method to search SNPs this part （5＇ UTR of SRPK1 in pig）, total of 40 Large White pigs were obtained as the research objects, but no polymorphism were found. Thus, 5＇ UTR of SRPK1 was speculated with a characteristic of high conservation, while it might have been directly or indirectly selected in commercial breeding. The paper provided a further feature of SRPK1 gene in molecular genetics.展开更多
通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3(serine/arginine-rich specific kinase 3,SRPK3)的全长基因CDS区...通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3(serine/arginine-rich specific kinase 3,SRPK3)的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR(Real-time)方法检测了SRPK3在1日龄和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS(1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A629→G629,T653→T653的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G1059→A1059,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。展开更多
基金Supported by 11th Five-year Plan Key Projects of National Science and Technology (2008BADB2B01)
文摘Part 5＇ UTR region of pig SRPK1 gene was cloned by inverse PCR （I-PCR）, then a 425 bp gene sequence was acquired. A promoter region in -1--309 bp was predicted by online tool （TFSEARCH） and 32 binding sites of transcription with scores higher than 85 were getten, of which scores of Spl, MyoD, and HSF2 were over 90. Some of these binding sites of transcription factors were connected with promoters, but TATA-box, which was important to gene expression, hadn＇t been found in this region. By using PCR-SSCP method to search SNPs this part （5＇ UTR of SRPK1 in pig）, total of 40 Large White pigs were obtained as the research objects, but no polymorphism were found. Thus, 5＇ UTR of SRPK1 was speculated with a characteristic of high conservation, while it might have been directly or indirectly selected in commercial breeding. The paper provided a further feature of SRPK1 gene in molecular genetics.
