Texel和乌珠穆沁绵羊妊娠中后期胎儿背最长肌中 AKT基因的发育表达模式

Abstract

Abstract: To select the best stable reference genes and investigate the developmental expression patterns of AKT gene in sheep fetal muscle. Quantitative RT-PCR was conducted for B2M, GAPDH, RPLPO, RPS18 and SDHC and also applied to investigate AKT mRNA expression at the gestation of 70 d, 85 d, 100 d, 120 d and 135 d in longissimus dorsi muscle of Texel and Ujumuqin sheep. The five reference genes of expression stability were analyzed by the GeNorm software, and the result showed that RPS18＞RPLPO＞SDHC＞B2M＞YWHAB. Furthermore, SDHC, RPLPO and RPS18 were identified as the most stable reference genes in the present study. In all the five stages, the expression patterns of AKT were dissimilar in the two sheep breeds. Strikingly, at the gestation of 85 d and 120 d, the expression of AKT reached their peak values in Texel,in contrast, in Ujumuqin it was at 100 d. These findings were consistent with our previous study which reflected the histological characteristics during the second half of gestation in the two sheep breeds, and this further verify that the surges in myofiber hyperplasia occurred at 85 d and 120 d in Texel sheep, whereas a unique proliferative surge appeared at 100 d in Ujumqin sheep. The results indicated that the transcript levels of AKT gene exhibited specific developmental expression changes between Texel and Ujumuqin sheep, and provided some data for further study on the effect of AKT gene.

Key words: Texel; Ujumuqin; AKT gene; quantitative RT-PCR; reference genes; developmental expression patterns

CLC Number:

Q786

ZHANG Xiao-ning, LU Jian, REN Hang-xing, WEI Cai-hong, ZHNAG Li, ZHAO Fu-ping, DU Li-xin. Developmental Expression Patterns of AKT Gene in Fetal Longissimus Dorsi Muscle between Texel and Ujumqin Sheep during the Second Half of Gestation[J]. .

References

1 陈烨,李凯,周宇荀,等. 小鼠基因转录表达分析中内参基因的优选[J]. 中国实验动物学报, 2011,19(3):197~202.

2 詹军芳. AKT-1和STAT-1介导乳腺癌始动细胞CD44⁺/CD24^-/low辐射抵抗的相关性研究.广州:南方医科大学,2011.

3 Amirouche A, Durieux A C, Banzet S, et al. Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle[J]. Endocrinology, 2009, 150:286~294.

4 Brunet A, Bonni A, Zigmond M J, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor[J]. Cell, 1999, 96:857~868.

5 Bustin S A, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction [J]. J Biomol Tech, 2004, 15:155~166.

6 Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J]. Nat Genet, 2006, 38:813~818.

7 Frost R A, Lang C H .Protein kinase B/Akt: a nexus of growth factor and cytokine signaling in determining muscle mass[J]. Appl Physiol, 2007, 103:378~387.

8 Hellemans J, Mortier G, De Paepe A, et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[J]. Genome Biol, 2007, 8:R19.

9 Ji M, Zhang Q, Ye J, et al. Myostatin induces p300 degradation to silence cyclin D1 expression through the PI3K/PTEN/Akt pathway[J]. Cell Signal, 2008, 20:1452~1458.

10Li Z B, Kollias H D, Wagner K R. Myostatin directly regulates skeletal muscle fibrosis[J]. Biol Chem, 2008, 283:19371~19378.

11Liu D W, Chen S T, Liu H P. Choice of endogenous control for gene expression in nonsmall cell lung cancer[J]. Eur Respir, 2005,26:1002~1008.

12Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta delta C(T)) method[J]. Methods,2001,25:402~408.

13Lokireddy S, Mouly V, Butler-Browne G, et al. Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins[J]. Am J Physiol Cell Physiol,2011,301(60):C1316~C1324.

14Morissette M R, Cook S A, Buranasombati C, et al. Myostatin inhibits IGF-Ⅰ-induced myotube hypertrophy through Akt[J]. Am J Physiol Cell Physiol, 2009, 297:C1124~C1132.

15Olsvik P A, Lie K K, Jordal A E, et al. Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon[J]. BMC Mol Biol, 2005, 6(21):112~118.

16Ren H, Li L, Su H, et al. Histological and transcriptome-wide level characteristics of fetal myofiber hyperplasia during the second half of gestation in Texel and Ujumqin sheep[J]. BMC Genomics, 2011, 12(4):11~16.

17Rennie M J, Wackerhage H, Spangenburg E E. Control of the size of the human muscle mass[J]. Annu Rev Physiol, 2004, 66:799~828.

18Rommel C, Clarke B A, Zimmermann S, et al. Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt[J]. Science, 1999, 286:1738~1741.

19Schmid H, Cohen C D, Henger A, et al. Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies[J]. Kidney Int, 2003, 64:356~360.

20Schmittgen T D, Zakrajsek B A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR[J]. Biochem Biophys Methods, 2000, 46:69~81.

21Selvey S, Thompson E W, Matthaei K, et al. Beta-actin--an unsuitable internal control for RT-PCR[J]. Mol Cell Probes, 2001, 15:307~311.

22Trendelenburg A U, Meyer A, Rohner D, et al. Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size[J]. Am J Physiol Cell Physiol,2009,296:C1258~C1270.

23Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7):RESEARCH0034.

24Vary T C, Lynch C J. Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation[J]. Am J Physiol Endocrinol Metab, 2006, 29:631~642.

25Yang W, Zhang Y, Li Y, et al. Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3 beta pathway and is antagonized by insulin-like growth factor 1[J]. Biol Chem, 2007, 282:3799~3808.

26Zhu G, Chang Y, Zuo J, et al. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH [J]. Biochem Biophys Res Commun, 2001, 281:951~956.

[1]	LI Bin, REN Tingting, QIU Meiyu, LI Zhiqiang, YAN Xiaofei, HAN Bing, LIU Mingjun, HE Sangang, YIMING·Sulaiman. Paternity Identification of Texel×Kazakh Sheep Based on Microsatellite Multiple PCR Technology [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2171-2180.
[2]	SHENG Hui, ZHANG Linlin, GUO Yiwen, LI Xin, DING Xiangbin, GUO Hong. A Selection of Reference Genes for Real-Time Quantitative PCR in Bovine Skeletal Muscle Satellite Cells Interfering with MSTN [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 381-391.
[3]	ZHANG Rong, SHI Xiao-li, CHEN Chao-gui, WEN Guang-lin, ZHAO Zhong-hai. Screening of Reference Genes for Gene Expression in Layer Hens Tissues Using Real-time Quantitative PCR [J]. , 2017, 44(8): 2226-2233.
[4]	ZHAO Ling-na, JIN Hong-yan, LIANG Lin, LI Gang. A SYBR Green Ⅰ Based on Real-time Quantitative RT-PCR Assay for Specific Detection of Peste des Petits Ruminants Virus [J]. , 2016, 43(11): 2844-2851.
[5]	ZHANG Ran-ran, LIU Hua-miao, XING Xiu-mei, HU Da-yong. Selection and Validation of Reference Genes in Velvet Antlers Tissues [J]. , 2015, 42(4): 883-889.
[6]	WANG Feng-xue, HUANG Shuang, LIU Ying, YANG Yong, SUN Na, ZHU Hong-wei, ZHANG Shu-qin, CHENG Shi-peng, WEN Yong-jun. Establishment and Application of Real-time Fluorescent Quantitative RT-PCR Method of the Detection of Three Swine Cytokines [J]. , 2015, 42(1): 24-31.
[7]	WANG Rong, LI Wen-wen, WANG Yan, LIU Ya-gang, YU Qiong, REN Yong-gang. Establishment and Application of Real-time Fluorescent Quantitative RT-PCR Method for Detection of Bovine Viral Diarrhea Virus [J]. , 2014, 41(2): 35-39.
[8]	LIU Hao-peng, HU Jing-jing, TANG Xu, PEI Zhang-fu, LI Bing, LI Lin, CHEN Rui-ai, HE Dong-sheng. Development and Application of One-step Real-time Fluorescent Quantitative RT-PCR Assay for Rapid Detection of H1 Subtype Swine Influenza Virus [J]. , 2014, 41(11): 63-68.
[9]	WANG Jian-zhong. Development of SYBR Green Ⅰ Fluorescence Quantitative RT-PCR Assay for Detection of Transmissible Gastroenteritis Virus of Swine S Gene [J]. , 2014, 41(1): 66-71.
[10]	ZHANG Shi-fang, WEI Cai-hong, LU Jian, ZHANG Xiao-ning, ZHOU Xin-lei, ZHANG Shu-zhen, WANG Guang-kai, CAO Jia-xue, ZHAO Fu-ping, ZHANG Li, DU Li-xin. Identification of the microRNAome in Texel Sheep by Deep Sequencing [J]. , 2013, 40(9): 19-22.
[11]	WU Qiao, ZHANG Bin, TANG Cheng, YUE Hua. Selection of Reference Genes in Mice Infected with H5N1 Avian Influenza Virus [J]. , 2013, 40(9): 55-60.
[12]	SONG Jia-zhe, SUN Fu-bo, ZHUANG Kai-ge, XUE Kai. Real-time Quantitative RT-PCR Analysis of Animal miRNA Expression [J]. , 2013, 40(9): 109-113.
[13]	LIU Ji-yu, DU Yi-jun, LIU Xing, WU Jia-qiang, LI Jun, CONG Xiao-yan, ZHAO Xin-hua, XU Shao-jian, SUN Wen-bo, SHI Jian-li, SHAN Hu, WANG Jin-bao. Establishment and Preliminary Application of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Type Ⅰ Interferon Effect Factor of HeLa Cells [J]. , 2013, (5): 1-7.
[14]	KONG Gang-rui, WU Zhi-ming, YAN Ruo-qian, ZHAO Ming-jun, WANG Dong-fang, ZHAO Xue-li, YAN Zhi-ling, CHENG Jun-zhen, CHEN Hui-juan, JIN Li-fen. Establishment and Application of Duplex Real-time Fluorescent Quantitative RT-PCR Assay for Identification of Highly and Lowly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus [J]. , 2013, 40(12): 27-33.
[15]	ZHOU Hong-miao, YUE Hua, TANG Cheng, WU Qiao. Selection of Stable Reference Genes in Mice Incubated with LPS Stimulation [J]. , 2013, 40(10): 68-72.

Developmental Expression Patterns of AKT Gene in Fetal Longissimus Dorsi Muscle between Texel and Ujumqin Sheep during the Second Half of Gestation

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments