绵羊YAP1基因的克隆及其真核表达载体和突变型载体的构建

doi:10.11843/j.issn.0366-6964.2013.08.004

Abstract

Abstract:

The study was conducted to clone sheep YAP1 CDS and construct a eukaryotic expression plasmid and a mutant that could not be phosphorylated at Ser42. The CDS of sheep YAP1 was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, and was cloned into pMD19-T simple vector by T/A Ligation to sequence the DNA fragment. After double digestion, YAP1 coding region was subcloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR，restriction endonucleases analysis and sequencing were used to conform both of the recombinant plasmids. The sheep full-length YAP1 cDNA sequence was 1 712 bp in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine at 42^ed amino acid by PCR，restriction digestion and sequencing. The results showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, which paved the foundation for further studies on the YAP1 protein expression and its biological activities.

CLC Number:

S826
S813.3

LI Da,SUN Wei, SU Rui, ZHANG Zhan-ying, MA Yue-hui, ZHANG You-fa,CHEN Ling, WU Wen-zhong, ZHOU Hong. Molecular Cloning and Construction of Eukaryotic Expression Vector and Mutant Vector of Sheep YAP1 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1198-1204.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2013.08.004

https://www.xmsyxb.com/EN/Y2013/V44/I8/1198

References

［1］ZHAO B,LI L,LEI Q Y,et al.The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version［J］. Genes Dev,2010,24:862-874.

［2］SUDOL M, HARVEY K F.Modularity in the Hippo signaling pathway［J］.Trends Biochem Sci,2010,35(11):627-633.

［3］YIN M X, ZHANG L. Hippo signaling:A hub of growth control, tumor suppression and pluripotency maintenance［J］.J Genet Genomics,2011,38:471-481.

［4］周洲，朱金水.Hippo 通路及YAP基因研究的新进展［J］.现代肿瘤医学，2011,19（6）：1241-1244.

［5］GISE A V,LIN Z Q,SCHLEGELMILCH K,et al. YAP1,the nuclear target of Hippo signaling,stimulates heart growth through cardiomyocyte proliferation but not hypertrophy［J］.PNAS, 2012,109(7):2394-2399.

［6］SCHLEGELMICH K, MOHSENI M, KIRAK O, et al. Yap1 Act downstream of α-catenin to control epidermal proliferation［J］.Cell, 2011, 144(5):782-795.

［7］WANG W Q,HUANG J,CHEN J J.Angiomotin-like proteins associate with and negatively regulate YAP1［J］.J Biol Chem,2010,286:4364-4370.

［8］FERNANDEZ L A,NORTHCOTT P A,DALTON J,et al.YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation［J］.Current Issue,2009,23:2729-2741.

［9］YOKOYAMA T,OSADA H, MURAKAMI H, et al. YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation［J］.Carcinogenesis,2008,29(11):2139-2146.

［10］ZHANG J M, JI J Y,YU M, et al. YAP-dependent induction of am-phiregulin identifies a non-cell-autonomous component of the Hippo pathway［J］.Nat Cell Biol，2009,11(12):1444-1450.

［11］李宁,肖虹,郑绘霞,等.YAP蛋白在膀胱尿路上皮肿瘤中的表达及临床意义［J］.山西医科大学学报，2011,42(5): 383-386.

［12］张靖,朱金水.Hippo-YAP信号通路与胃癌相关性研究［J］.国际消化病杂志,2011,31(4):207-215.

［13］赵修民，王德林.YAP基因表达与肿瘤的关系［J］.广东医学,2011,32(12):1628-1629.

［14］刘丛丛,梁建芳,郑绘霞,等.YAP在结直肠腺癌组织中的表达及意义［J］.中国药物与临床,2011,11（1）：23-26.

［15］刘玉，夏立平，郑武平.YAP在乳腺癌中的表达及其临床意义［J］.海南医学院学报,2010,16(6):699-704.

［16］ZHAO B, LEI Q Y, GUAN K L.The Hippo-YAP pathway:new connections between regulation of organ size and cancer［J］.Curr Opin Cell Biol,2008,20:638-646.

［17］WANG K,DEGERNY C,XU M,et al.YAP, TAZ, and Yorkie: a conserved family of signal-responsive transcriptional coregulators in animal development and human disease［J］. Biochem Cell Biol，2009，87：77-91.

［18］BASU S,TOTTY N F, IRWIN M S, et al.Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis［J］.Mol Cell，2003，11：11-23.

［19］ZHAO B, YE X, YU J, et al. TEAD mediates YAP-dependent gene induction and growth control［J］. Genes Dev，2008，22: 1962-1971.

［20］CAMARGO F D, GOKHALE S, JOHNNIDIS J B,et al. YAP1 increases organ size and expands undifferentiated progenitor cells［J］. Curr Biol，2007，17：2054-2060.

［21］HUDSON N J, REVERTER A, DALRYMPLE B P.A differential wiring analysis of expression data correctly identifies the gene containing the causal mutation［J］. PLoS Comp Biol, 2009, 5(5):e1000382.

［22］HUDSON N J, REVERTER A, WANG Y, et al.Inferring the transcriptional landscape of bovine skeletal muscle by integrating co-expression networks［J］. PLoS ONE,2009, 4(10):e7249.

［23］KIM H S, LIANG L, DEAN R G,et al. Inhibition of preadipocyte differentiation by Myostatin treatment in 3T3-L1 cuLtures［J］.Biochem Biophys Res Commun,2001,281(4):902-906.

［24］GARIKIPATI D K, GAHR S A, ROALSON E H,et al. Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a &-2b): genomic organization, differential expression and psuedogenization［J］.Endocrinology,2007,148(5):2106-2115.

［25］HENMMINGS K M,PARR T, DANIELZ C T R,et al. Examination of myosin heavy chain isoform expression in ovine skeletal muscles［J］. Am Soc Anim Sci, 2009,87: 3915-3922.

［26］FERRIGNO O, LALLEMAND F, VERRECCHIA F,et al.Yes-associated protein(YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-β/Smad signaling［J］.Oncogene, 2002,32:4879-4884.

［27］WATT K I, JUDON R, MEDLOW P,et al.Yap is a novel regulator of C2C12 myogenesis［J］.Biochem Biophy Res Commun, 2010,393:619-624.

［28］鞠会艳，夏咸柱，高玉伟，等.犬瘟热病毒基因疫苗表达载体构建及在真核细胞中的表达［J］.东北农业大学学报，2009,40(10):85-90.

[1]	WANG Haibo, ZHAN Jinshun, GU Zhiyong, CHEN Xinfeng, PAN Yue, JIA Haobin, ZHONG Xiaojun, LI Kairong, ZHAO Shengguo, HUO Junhong. Comparative Study on Meat Quality Characteristics of Three-Way Hybrid Sheep Charolais×Duper×Hu and Charolais×Australian White×Hu and Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 110-119.
[2]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[3]	SONG Pengyan, WANG Siwei, YUE Qiaoxian, ZHANG Yinliang, CHEN Xiaoyong, ZHOU Rongyan. Identification of oar-miR-200b Promoter and Effects on Mitochondrial Function in Follicular Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5066-5076.
[4]	HAN Haoyuan, LI Shikai, YANG Ruiqiao, LI Manman, LI Jun, HA Si, ZHAO Jinyan, WEI Hongfang, QUAN Kai. Mining Key Candidate Genes for High Reproduction Performance of Huai Goats Based on Transcriptome Sequencing [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5077-5090.
[5]	CHEN Conghui, DUAN Chunhui, YANG Xinyu, XIA Cui, GUO Yunxia, JI Shoukun, YAN Hui, LIU Yueqin, ZHANG Yingjie. Effects of Different Treatments of Transportation Stress on Energy Metabolism of Lambs Muscle after Slaughter [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5101-5111.
[6]	LI Dengpan, MA Keyan, HAN Jintao, BAI Yaqin, LI Taotao, MA Youji. Detection and Analysis of Whole Genome Selection Signal of Yongdeng Qishan Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4577-4588.
[7]	ZHAO Xueyang, LI Danni, WANG Yuchen, GUO Lei, WANG Li, HUANG Jie, JIAO Yiqiang, AN Xiaopeng, ZHANG Xiyun, ZHANG Lei, SONG Yuxuan. GWAS Analysis of Lambing Traits in East Friesian and Hu Crossbred Sheep and Verification of Candidate Gene GRID2 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4625-4635.
[8]	ZHANG Hanyue, ZHAO Dan, LIANG Yu, ZHAO Bishi, FAN Mengdan, QIAO Liying, LIU Jianhua, YANG Kaijie, PAN Yangyang, LIU Wenzhong. miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3262-3274.
[9]	LIU Linli, PENG Xuelan, LI Bo, CHENG Lefan, CIREN Lamu, ZHANG Enping. Effect of Overexpression of UCP3 Gene on the Differentiation of Sahu Sheep Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3275-3285.
[10]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.
[11]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[12]	LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.
[13]	MA Youji, CHEN Pengfei, MA Qing, WU Yi. Effects of Different Exercise Amounts on Rumen Microflora Diversity of Tan Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3393-3405.
[14]	WANG Wannian, CHEN Sijia, GAO Jinrong, WEN Zhonghao, YUAN Mengjiao, ZHANG Hongzhi, PANG Zhixu, QIAO Liying, LIU Wenzhong. Simulation Study on Genomic Selection of Sex-limited Traits Using Multilayer Perceptron in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2824-2835.
[15]	XIONG Chengkun, ZHANG Daoliang, YANG Yue, DING Hongyan, ZHAO Jie, LI Yu, WANG Xichun, FENG Shibin, ZHAO Chang, TANG Jishun, WU Jinjie. Effect of Rutin on Rumen Fermentation, Rumen Flora Structure and Antioxidant Properties in Perinatal Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2898-2909.

Molecular Cloning and Construction of Eukaryotic Expression Vector and Mutant Vector of Sheep YAP1 Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments