猪BLM基因N端、C端的克隆及其在不同组织与疾病中的表达分析

中国畜牧兽医2018，45(8) :207,-2085China Animal Husbandry& Veterinary Medicinedoi：10. 16431/j. cnki. 1671-7236. 2018. 08. 005猪B LM基因N端、C端的克隆及其在不同组织与疾病中的表达分析王赛楠123，许厚强1!，3"，赵佳福123(1.贵州大学，高原山地动物遗传育种与繁殖教育部重点实验室，贵阳550025 &2.贵州省动物遗传育种与繁殖重点实验室，贵阳550025 ;3.贵州大学生命科学学院，贵阳550025)摘要：为探究猪B LM解旋酶N端、C端的结构功能及其在不同组织和疾病中的相对表达量，本研究采用RT-PCR技术扩增从江香猪B LM基因N端和C端，利用生物信息学软件对克隆所获序列进行分析，同时采用实时荧光定量P=R技术分析B L M基因在猪不同组织及病样中的表达差异。

结果显示，试验成功克隆了从江香猪B LM基因N端1 025 b p和C端1 300 b p序列，与GenBank中猪B LM基因序列（登录号：NM—001123084.1)同源性为100P生物信息学软件预测显示，猪B LM基因序列长度为4 316bP，包含4 280 bP的开放阅读框，编码一个由1 426个氨基酸组成的多肽链；氨基酸序列比对发现，猪B LM基因氨基酸序列与牛的同源性最高（85%);结构域预测表明，猪B LM基因有DEXDc、HELICc、R Q C和H R D C4个结构域；系统进化树分析表明，猪B LM基因与牛亲缘关系最近；实时荧光定量PC R结果显示，以心脏为对照，B LM基因在从江香猪各组织中均有表达，其中在睾丸中的相对表达量最高，其次为肺脏、肝脏、脾脏、心脏、肾脏、小肠和大肠，在睾丸和肺脏中的表达量均极显著高于其他组织（P<0.01#B L M基因在患繁殖与呼吸道综合征猪肺脏、患白血病鸡肝脏和患鸭瘟鸭肝脏中的表达水平均极显著或显著高于正常组织（P<0.01P<0. 05#提示B LM基因的变化可能也会导致动物疾病的发生’本试验结果可为后续畜禽BLM解旋酶原核表达载体的构建及其在重大疾病的发生和预防等研究提供理论依据。

关键词：猪；B LM基因；序列分析；组织表达中图分类号：Q78 文献标识码：A 文章编号：1671-7236(2018)08-2076-10Cloning of N-terminal and C-terminal of Pig BLM Gene andIts Expression Analysis in Different Tissues and DiseasesW ANG Sainan1，2,3，XU H ouqiang1，2,3"，ZH A O Jiafu1，2,3(1. Key Laboratory o f Animal Genetics，Breeding and Reproduction in the Plateau Mountain Region，Ministry o f Education，Guizhou University，Guiyang550025? China; 2. Key Laboratory o fAnimal Genetics，Breeding and Reproduction in Guizhou Province，Guiyang550025, China &3. College o f L ife Sciences，Guizhou University，Guiyang550025, China)A bstract：In order to investigate the structure and function of N-term inal aBLM gene and its relative expression in different tissues and diseases,the N-term inal and C-term i­nal of BLM gene from Congjiang Xiang pig w ere am plified by R T-PC R m ethod in this study, thesequence of clones was analyzed using bioinform atics softw ares. A t the same tim e, the expressionof BLM gene in different tissues and diseases of pig was analyzed by Real-tim e quantitative PCR.T he results showed t hat the N-term inal (1 025 bp) and C-term inal (1 300 bp) sequences of BLMgene from Congjiang Xiang pig w ere successfully cloned, and the sequence hom ology w ith Gen-收稿日期：2018-04-08基金项目：国家自然科学基金（700703131123);教育部中国与美大、欧洲地区教育科研合作项目（教财司函[2017*60号）作者筒介：王赛楠（1293-)，女，陕西渭南人，硕士生，研究方向：细胞生物学，E-mail: 1838261858®qq. com"通信作者：许厚强（1257-)，博士，教授，研究方向：细胞分子生物学，E-mail :gzdxxhq® 163. com8期王赛楠等：猪B LM基因N端、C端的克隆及其在不同组织与疾病中的表达分析Bank (accession No. ： NM_001123084. 1) was 100% ；T he bioinform atics prediction results showed that the sequence l ength ff pig BLM gene was 4 316 bp, including an open reading fram e of 4 280 bp, and the corresponding polypeptide was consisted ff 1 426 amino acids. Amino acid se­quence com parison resutt s howed that the amino acid sequence of pig BLM gene was the highest hom ology w ith cow (85 %). T he phylogenetic tree analysis showed that the pig BLM helicase had four genetic domains of DEXDc , H ELICc , RQC and H R D C； Phylogenetic tree analysis showed that the BLM gene relationship betw een pig and bovine was the closest ； Real-tim e quantitative PCR result showed that com pared w ith the heart tissu e,th e expression of BLM gene in Congjiang Xiang pig was detected i n all tissu es, there was the highest relative expression in te stis, followed by the tissues of lun g, liver, spleen, h e a rt, kidney, small intestine and large intestine. T he expres­sion levels in testis and lung w ere extrem ely significantly higher than those in other tissues (P<0. 01). T he expression of BLM gene in lung of porcine reproductive and respiratory syn­drom e, liver of chicken leukem ia and quail duck was extrem ely significantly or significantly higher than that in norm al tissues (P<0. 01 ；P<0. 05). T he difference indicated that the change in BLM helicase m ight also lead to animal diseases. T he results provided a theoretical basis for construc­tion of prokaryotic expression vector of livestock and poultry BLM helicase and their occurrence and prevention in m ajor diseases.Key w ords：p ig；B LM gene；sequence analysis；tissue expressionB loom解旋酶（BLM)是R e cQ家族D N A解旋 酶中的重要成员，B L M基因突变会使B L M解旋酶 活性降低，D N A修复过程受阻，导致染色体不稳定，进而引起 Bloom 综合征（B loom syndrom e，BS)[1]的发生，患者免疫功能低下，易患白血病和其他恶性肿 瘤，这一现象引起了人们的广泛关注。

人B L M基 因位于15q26. 1区带上[2]，包含1个长度为4 254 b p的开放阅读框，编码1 417个氨基酸残基组 成的多肽链，分子质量约为159 ku[]。

B L M解旋酶 主要由N端、解旋酶核心结构域（helicase core)4—5]、RQC 结构域（RecQ C-term inal do­m ain)[6]、HRDC 结构域（helicase and RNaseD C- term inal dom ain)[7]和位于C端的核定位信号区(nuclear localization signal，N LS)组成。