猪胰岛素启动子序列分析及调控基因在胰腺组织特异性表达验证

doi:10.11843/j.issn.0366-6964.2013.10.002

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (10): 1516-1521.doi: 10.11843/j.issn.0366-6964.2013.10.002

猪胰岛素启动子序列分析及调控基因在胰腺组织特异性表达验证

胡炳俊，王超群，阮进学，辛磊磊，牟玉莲，杨述林^*，李奎

（中国农业科学院北京畜牧兽医研究所，农业部畜禽遗传资源与种质创新重点实验室，北京 100193）

收稿日期:2013-03-29 出版日期:2013-10-23 发布日期:2013-10-23
通讯作者: 副研究员，E-mail: hi_ysl@yahoo.com.cn
作者简介:胡炳俊（1987-），男，安徽桐城人，硕士生，主要从事细胞工程与基因工程研究，E-mail: hubingjun888@163.com
基金资助:
国家科技支撑计划（2012BAI39B04；2011BAI15B02）；“863”计划（SQ2011AAJY2795）；“973”计划（2011CBA01005）；国家转基因生物新品种培育重大专项（2011ZX08010-003）

Analysis of the Porcine Insulin Promoter and Verification of the Regulatory Genes Specific Expression in Pancreatic Tissue

HU Bing-jun, WANG Chao-qun, RUAN Jin-xue, XIN Lei-lei, MOU Yu-lian,YANG Shu-lin^*, LI Kui

(Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193，China)

Received:2013-03-29 Online:2013-10-23 Published:2013-10-23

摘要/Abstract

摘要：

通过对不同中国地方猪品种胰岛素基因启动子序列差异分析，可为转基因调控我国小型猪胰腺β细胞功能研究和糖尿病模型制备提供依据。本研究利用PCR从通城猪、广西巴马小型猪和五指山小型猪3个品种猪基因组DNA中扩增猪胰岛素启动子全长，经比对分析获得具有地方猪品种特征的序列，在小鼠胰腺瘤细胞和转基因鼠中验证该序列调控目的基因表达的有效性和特异性。3个地方猪品种间1.5 kb长的胰岛素启动子序列无碱基差异，与国外猪品种仅有3个碱基差异；双荧光素酶报告基因检测系统证明该启动子能在胰腺瘤细胞高效启动下游基因表达；转CHOP和UCP2基因小鼠中，2个目的基因在胰腺组织中表达量显著高于肝脏、背肌和肾脏，且胰腺组织中蛋白丰度显著增加。本研究结果表明，从中国地方猪品种中分离的胰岛素启动子能够特异性调控目的基因在胰腺组织中表达，为转基因调控猪β细胞功能的动物模型建立奠定了基础。

Abstract:

The analysis of different Chinese local pig breeds insulin promoter sequence provided the basis for studying miniature pig β cell function and preparing diabetes model. In this study, porcine insulin promoters（PIP）from three pig breeds(Tongcheng pigs, Guangxi Bama Miniature pigs and Wuzhishan Miniature pigs) had been amplified by PCR. By the comparative analysis, the sequences of local pig breeds characteristics were got. To verify the validities and specificities of the gene expression regulatory of the sequence, pancreatic tumor cells and transgenic mice were used. There were no differences among the three local pig breeds in 1.5 kb insulin promoter sequences. Compared with the sequence from foreign pig breeds, there were only three nucleotide differences. Dual luciferase reporter gene detection system proved that the promoter could efficiently start downstream gene expression; In transgenic mice with CHOP and UCP2, two target genes expressions in pancreatic tissue were significantly higher than that in the liver, back muscles and kidneys, and pancreas tissue protein abundance significantly increased. The result of this study shows that the insulin promoter of Chinese local pig breeds can regulate target gene expression in the pancreatic tissue, which lays the foundation for producing transgenic pig model of regulating β cell function.

中图分类号:

S828
S813.3

胡炳俊，王超群，阮进学，辛磊磊，牟玉莲，杨述林，李奎. 猪胰岛素启动子序列分析及调控基因在胰腺组织特异性表达验证[J]. 畜牧兽医学报, 2013, 44(10): 1516-1521.

HU Bing-jun, WANG Chao-qun, RUAN Jin-xue, XIN Lei-lei, MOU Yu-lian,YANG Shu-lin, LI Kui. Analysis of the Porcine Insulin Promoter and Verification of the Regulatory Genes Specific Expression in Pancreatic Tissue[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1516-1521.

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参考文献

［1］YANG W, LU J, WENG J, et al. China national diabetes and metabolic disorders study group. Prevalence of diabetes among men and women in china ［J］. N Engl J Med, 2010, 362(12): 1090-1101.

［2］DAHL U, SJODIN A, SEMB H, et al. Cadherins regulate aggregation of pancreatic beta-cells in vivo ［J］. Development, 1996, 122: 2895-2902.

［3］HANAHAN D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes ［J］. Nature, 1985, 315: 115-122.

［4］VASAVADA R C, CAVALIERE C, D’ERCOLE A J, et al. Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia ［J］. Biol Chem, 1996, 271: 1200-1208.

［5］HARA M, WANG X, KAWAMURA T, et al. Transgenic mice with green fluorescent protein-labeled pancreatic beta-cells ［J］. Am J Physiol Endocrinol Metab, 2003, 284: 177-183.

［6］HOTTA M, TASHIRO F, IKEGAMI H, et al. Pancreatic beta cell-specific expression of thioredoxin, an antioxidative and antiapoptotic protein, prevents autoimmune and streptozotocin-induced diabetes ［J］. Exp Med, 1998, 188: 1445-1451.

［7］KRAKOWSKI M L, KRITZIK M R, JONES E M, et al. Transgenic expression of epidermal growth factor and keratinocyte growth factor in beta-cells results in substantial morphological changes ［J］. J Endocrinol, 1999, 162: 167-175.

［8］MARTIN J, HUNT S L, DUBUS P, et al. Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number ［J］.Oncogene, 2003, 22: 5261-5269.

［9］SPURLOCK M E, GABLER N K. The development of porcine models of obesity and the metabolic syndrome ［J］. J Nutr, 2008, 138: 397-402.

［10］LONDRIGAN S L, BRADY J L, SUTHERLAND R M, et al. Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets ［J］. Xenotransplantation, 2007,14: 119-125.

［11］袁小清，马向华，沈捷.核转录因子CHOP的研究进展［J］. 医学研究杂志，2008，8（37）: 15-18.

［12］周辉，张旭家.线粒体解偶联蛋白UCP2的研究进展［J］. 生命科学，2008，4（20）: 549-559.

［13］KHOI C, GAILLARD N E, TSAI M J. Beta2 and pancreatic islet development ［J］. En Rev, 2001, 56(1): 23-46.

［14］YANG K C, QI Z, YANAI G, et al. Cell coupling regulates Ins1, Pdx-1 and MafA to promote insulin secretion in mouse pancreatic beta cells ［J］. Process Biochem, 2011, 46: 1853-1860.

［15］MELLOUL D, MARSHAK S, CERASI E. Regulation of insulin gene transcription ［J］. Diabetologia March, 2002, 45(3): 309-326.

［16］BOAM D, CLARK A, DOCHERTY K. Positive and negative regulation of the insulin gene by multiple trans-acting factors［J］. Biol Chem, 1990, 265: 8285-8296.

［17］GERMAN M, RUTTER W J. The insulin gene promoter: a simplified nomenclature ［J］. Diabetes, 1995, 44(8): 1002-1004.

［18］WATANABE M, UMEYAMA K, KAWANO H, et al. The production of a diabetic mouse using constructs encoding porcine insulin promoter-driven mutant human hepatocyte nuclear factor-1alpha［J］. Reprod, 2007, 12(53): 189-200.

［19］MARJETA G, DAHLHOFF M, HERBACH N, et al. Specific transgene expression in mouse pancreatic β-cells under the control of the porcine insulin promoter ［J］. Mol Cell Endocrinol, 2010, 315(1-2): 219-224.

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猪胰岛素启动子序列分析及调控基因在胰腺组织特异性表达验证

Analysis of the Porcine Insulin Promoter and Verification of the Regulatory Genes Specific Expression in Pancreatic Tissue

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