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

中国畜牧兽医

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

张小宁¹, 陆健^1,2, 任航行³, 魏彩虹¹, 张莉¹, 赵福平¹, 杜立新¹

1. 中国农业科学院北京畜牧兽医研究所,北京 100193;
2. 扬州大学动物科学与技术学院,江苏扬州 225009;
3. 重庆市畜牧科学院,重庆 402460

收稿日期:2011-12-25 出版日期:2012-08-20 发布日期:2012-08-24
通讯作者: 杜立新 E-mail:lxdu@263.net
作者简介:张小宁(1986－),女,河南人,硕士,主要从事畜禽分子育种的研究。
基金资助:
国家十二五科技支撑计划(2011BAD28B05-2);国家自然基金(30972079)。

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

ZHANG Xiao-ning¹, LU Jian^1,2, REN Hang-xing³, WEI Cai-hong¹, ZHNAG Li¹, ZHAO Fu-ping¹, DU Li-xin¹

1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193,China;
2. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
3. Chongqing Academy of Animal Sciences, Chongqing 402460, China

Received:2011-12-25 Online:2012-08-20 Published:2012-08-24

摘要/Abstract

摘要： 选择绵羊胎儿期骨骼肌中表达稳定性高的内参基因,探讨AKT基因在绵羊妊娠中后期背最长肌中的发育性表达模式。以Texel和乌珠穆沁绵羊为研究对象,采用荧光定量PCR技术检测SDHC、YWHAB、RPLPO、RPS18、B2M 5个内参基因的mRNA表达水平,同时检测AKT基因mRNA丰度在其胎儿期70、85、100、120和135 d间的表达变化,并分析其在品种间和不同生长阶段间的表达差异及其对肌肉生长发育的影响。5个内参基因的定量结果经GeNorm程序统计学分析处理,表达稳定性由高到低排序为RPS18＞RPLPO＞SDHC＞B2M＞YWHAB,确定用RPLPO、RPS18和SDHC 3个基因校正绵羊胎儿期不同生长阶段的目的基因表达量。在5个发育阶段中,两个品种绵羊胎儿AKT基因表达模式有一定差异,AKT表达量在Texel绵羊85和120 d时均有明显的增加,而在乌珠穆沁绵羊的100 d时出现1个表达峰,这与试验对两品种绵羊组织学分析的结果是一致的,进一步印证了Texel绵羊在85和120 d以及乌珠穆沁绵羊在100 d时存在肌发生波的推测。以上结果初步揭示绵羊妊娠中后期生长发育过程中AKT基因表达的发育性变化模式和品种差异,为深入研究AKT基因的作用机制提供了理论基础。

关键词: Texel绵羊; 乌珠穆沁绵羊; AKT基因; 荧光定量PCR; 内参基因; 发育表达模式

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

中图分类号:

Q786

张小宁, 陆健, 任航行, 魏彩虹, 张莉, 赵福平, 杜立新. 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[J]. 中国畜牧兽医.

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]. .

参考文献

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]	麻宝艺, 盛陈艳, 刘宏莹, 马青霞, 汪婷婷, 李健明, 时坤, 杜锐, 冷雪. BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(6): 2326-2335.
[2]	吕晓楠, 徐立蒲, 张文, 王娜, 曹欢, 王小亮, 王姝, 王静波. 感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究[J]. 中国畜牧兽医, 2022, 49(3): 1077-1084.
[3]	张军芳, 孙建富, 孙斌, 唐琳, 王英, 王恩泽, 崔岩, 李强, 严昌国, 李香子. 延边牛PPARγ基因的克隆及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 2695-2704.
[4]	苗曼宁, 郭益文, 胡德宝, 曾雨晗, 李新, 张林林, 丁向彬, 郭宏. MSTN基因编辑牛肌肉转录组测序及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2271-2281.
[5]	李恒, 字向东. 牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析[J]. 中国畜牧兽医, 2021, 48(4): 1284-1293.
[6]	李建梅, 季正剑, 刘梅, 沈欣悦, 程旭, 俞燕, 戴亚斌. 藏鸡和隐性白羽鸡感染柔嫩艾美耳球虫后免疫相关基因的表达变化分析[J]. 中国畜牧兽医, 2021, 48(4): 1440-1448.
[7]	陈磊, 袁枫, 贺三刚, 玛依拉, 李文蓉. 绵羊FGF10基因克隆、序列分析及其在毛囊发育中的表达分析[J]. 中国畜牧兽医, 2021, 48(2): 425-432.
[8]	戴政列, 朱静静, 陈振宇, 周晓龙, 汪涵, 李向臣, 赵阿勇, 杨松柏. 猪SENP1基因克隆、生物信息学分析与表达研究[J]. 中国畜牧兽医, 2021, 48(12): 4382-4390.
[9]	姜懿轩, 王亚玲, 邢珊珊, 宋国华, 许会芬, 李明. 新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建[J]. 中国畜牧兽医, 2021, 48(10): 3501-3511.
[10]	李志强, 陶晨雨, 魏奥龙, 贾青. 猪性控精子纯度实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3682-3690.
[11]	王征帆, 朱利塞, 王娟, 李祎云, 向蕊, 应碧云, 王贵平, 贾爱卿, 白挨泉. PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用[J]. 中国畜牧兽医, 2021, 48(10): 3855-3863.
[12]	李安奇, 蒋蕾, 苏晓彤, 昝林森, 王洪宝. 秦川牛ACTC1基因的表达特性分析[J]. 中国畜牧兽医, 2020, 47(8): 2359-2367.
[13]	李京徽, 邢思远, 王希彩, 李庆贺, 赵桂苹, 张永宏, 文杰, 刘冉冉. 鸡肌内脂肪沉积相关候选基因筛选[J]. 中国畜牧兽医, 2020, 47(6): 1828-1836.
[14]	盛辉, 张林林, 郭益文, 李新, 丁向彬, 郭宏. 干扰MSTN的牛骨骼肌卫星细胞实时荧光定量PCR内参基因的筛选[J]. 中国畜牧兽医, 2020, 47(2): 381-391.
[15]	姜徽, 刘攀, 李媛媛, 刘奎国, 黄锡霞, 刘江卫, 胥磊, 杜建文, 桑扎根, 杨雪梅, 赵番番, 王丹. 新疆褐牛乳房炎抗性相关基因的表达及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(1): 90-97.