牦牛热休克蛋白70-12B基因(HSPA12B)的克隆及其在体外受精胚胎中的表达  

Cloning of Bos Grunniens Heat Shock 70 k D Protein- 12B Gene(HSPA12B) and Detection of Its Expression in Yak In vitro Fertilization Embryos

作　　者：吕鹏[1] 潘阳阳[1] 崔燕[1] 何翃闳 刘鹏刚[1] 李秦[1] 郑红飞[1] 樊江峰[1] 余四九[1] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2015年第9期1226-1233,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31472244)

摘　　要：为研究牦牛(Bos grunniens)热休克蛋白70-12B基因(heat shock 70 k D protein-12B,HSPA12B)的序列特性及其在体外受精胚胎中的表达情况,本研究根据黄牛(Bos taurus)HSPA12B基因序列5'端和3'端的保守序列设计特异性引物,RT-PCR扩增之后通过基因克隆得到HSPA12B基因(Gene Bank登录号:KR258751),利用生物学信息软件对其进行生物学信息分析,并采用实时定量PCR(quantitative Real-time PCR,q RT-PCR)检测其在牦牛体外受精胚胎2-4细胞期、5-8细胞期、桑葚期、囊胚期4个不同时期胚胎中的表达水平。生物学信息分析结果显示,牦牛HSPA12B基因序列具有一个长705 bp的编码区,可编码234个氨基酸,其中丙氨酸(Ala)含量最高(10.3%),天冬酰胺(Asn)含量最少(0.9%);同源性和系统进化树分析显示,牦牛HSPA12B基因与黄牛HSPA12B的同源性最高,为99.5%,表明HSPA12B在进化中具有高度保守性;编码产物无信号肽,为非跨膜蛋白,不属于分泌蛋白,整体表现为亲水性。q RT-PCR检测结果显示,HSPA12B在牦牛体外受精2-4细胞期胚胎、5-8细胞期胚胎、桑椹胚和囊胚中均有表达;HSPA12B在2-4细胞期胚胎中的相对表达量分别是在5-8细胞期胚胎、桑椹胚和囊胚中相对表达量的14.814 8、4.269 9和40.485 8倍;HSPA12B在2-4细胞期胚胎中的相对表达量,均与在5-8细胞期胚胎、桑椹胚和囊胚中的相对表达量差异性极显著(P<0.01)。本研究提示,HSPA12B在牦牛体外受精胚胎的发育过程中非常重要,可能起着抑制细胞凋亡,延缓细胞衰老,调控胚胎早期发育的作用。In order to study the sequence characteristics of heat shock 70 kD protein-12B gene （HSPA12B） and its expression in yak （Bos grunniens） early in vitro fertilization （IVF） embryos, the specific primers were designed by the conservative sequence of HSPA12B gene of the cattle （Bos taurus）, and the gene sequence could be obtained by cloning. The HSPA12B sequence was analyzed by bioinformatics. The HSPA12B in early IVF embryos were detected by the methods of quantitative Real-time PCR （qRT-PCR）. The results of bioinformatics showed the coding region of the yak HSPA12B gene was 705 bp in length, encoding 234 amino acids, and the content of Alanine resduces was the highest （about 10.3%） and the content of Asparagine resduces was the lowest （about 0.9%）. According to homology analysis, the similarity of HSPA12B gene in yak and cattle was up to 99.5%, showing the HSPA12B gene was highly conserved. The protein was hydrophilic and had no signal peptide, which was not a transmembrane and secreted protein, qRT-PCR results showed that the HSPA12B in yak early embryos fertilized in vitro expressed in each period, and the relative expression of HSPA12B in 2-4 cell stage embryos was 14.814 8, 4.269 9 and 40.485 8 folds respectively, compared to the 5-8 cell stage embryos, morulae and blastocysts. The difference of the relative expression of HSPA12B between 2-4 cell stage embryos and 5-8 cell stage embryos, morula and blastocysts was very significant （P〈0.01）. These results showed HSPA12B played an important role in the development of embryos IVF and can inhibit cell apoptosis, cell senescence, and regulate embryonic development.

关 键 词：牦牛 热休克蛋白70-12B基因(HSPA12B) 基因克隆 生物信息学分析 体外培养(IVF) 实时荧光定量PCR(qRT-PCR) 

分 类 号：S823[农业科学—畜牧学]