鸡微粒体甘油三酯转运蛋白样基因生物学特性及表达调控

doi:10.11843/j.issn.0366-6964.2022.09.008

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (9): 2900-2911.doi: 10.11843/j.issn.0366-6964.2022.09.008

鸡微粒体甘油三酯转运蛋白样基因生物学特性及表达调控

徐春林¹, 曹玉珠¹, 夏天¹, 贾其辉¹, 王丹丹¹, 郑航¹, 田亚东^1,2,3, 康相涛^1,2,3, 蒋瑞瑞^1,2,3, 刘小军^1,2,3*, 李红^1,2,3*

1. 河南农业大学动物科技学院, 郑州 450046;
2. 河南省家禽育种国际联合实验室, 郑州 450046;
3. 河南省鸡种质资源创新与利用重点实验室, 郑州 450046

收稿日期:2022-01-12 出版日期:2022-09-23 发布日期:2022-09-23
通讯作者: 李红,主要从事家禽遗传育种研究,E-mail:lihong19871202@163.com;刘小军,主要从事动物遗传育种研究,E-mail:xjliu2008@hotmail.com
作者简介:徐春林(1990-),男,河南信阳人,硕士生,主要从事家禽遗传育种研究,E-mail:503479889@qq.com
基金资助:
国家自然科学基金青年基金项目（31902146）；河南省高等学校重点科研项目（21A230011）；河南省高校科技创新团队支持计划（21IRTSTHN022）

Biological Characteristics and Expression Regulation of Chicken Microsomal Triglyceride Transfer Protein Like Gene

XU Chunlin¹, CAO Yuzhu¹, XIA Tian¹, JIA Qihui¹, WANG Dandan¹, ZHENG Hang¹, TIAN Yadong^1,2,3, KANG Xiangtao^1,2,3, JIANG Ruirui^1,2,3, LIU Xiaojun^1,2,3*, LI Hong^1,2,3*

1. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China;
2. International Joint Research Laboratory for Poultry Breeding of Henan, Zhengzhou 450046, China;
3. Henan Key Laboratory for Innovation and Utilization of Chicken Germplasm Resources, Zhengzhou 450046, China

Received:2022-01-12 Online:2022-09-23 Published:2022-09-23

摘要/Abstract

摘要： 旨在探究鸡微粒体甘油三酯转运蛋白样基因（MTTPL）生物学特性及其表达调控机制，为进一步探讨其在鸡肝脂质代谢中的生物学功能奠定基础。本研究首先采用PCR和测序技术，克隆MTTPL cDNA序列；利用在线软件对MTTPL蛋白结构域、三维空间结构及系统进化树进行分析；构建pcDNA3.1-MTTPL-EGFP过表达载体，通过与内质网标签蛋白的表达载体DsRed-λ共转染鸡肝癌细胞系（LMH）细胞，对MTTPL进行亚细胞定位；分别采集不同周龄卢氏绿壳蛋鸡（1日龄、1周龄、10周龄、30周龄每个周龄各8只）组织样，采用荧光定量PCR分析MTTPL基因和鸡微粒体甘油三酯转运蛋白基因（MTTP）的时空表达谱；然后用0.5、1和2 mg·kg^-1体重浓度的17 β-雌二醇分别处理海兰褐鸡（每组20只）12和24 h后，分析雌激素对肝MTTPL和MTTP表达的影响；最后用1、50、和100 nmol·L^-1 17β-雌二醇及雌激素受体拮抗剂分别处理鸡胚肝原代细胞12 h （每组3个重复），研究雌激素调控MTTPL基因表达的作用机制。结果表明，鸡MTTPL的CDS区全长为2 646 bp，可编码881个氨基酸，与人和鸡MTTP拥有共同的祖先；定位于细胞内质网；鸡MTTPL和MTTP具有与人MTTP相同的功能域，且三维结构相似度偏差较小，分别为0.090和0.064；MTTPL基因在鸡肝和肾组织中高表达，MTTP在鸡肝、肾和小肠中高表达；在肝中，MTTPL和ApoB的表达水平随周龄的增加均显著上升（P<0.05），而MTTP的表达仅在10周前随周龄增加显著升高（P<0.05），而在产蛋前（10周龄）和产蛋期（30周龄）无显著变化（P>0.05）；在17β-雌二醇处理鸡12和24 h时后，MTTPL及ApoB在肝中均显著上调表达（P<0.05），而MTTP在高浓度处理时表达量显著下调（P<0.05），在低浓度时无显著变化；与对照组相比，雌激素可显著上调鸡胚肝原代细胞中ApoB和MTTPL的表达（P<0.05），而MTTP表达水平无显著变化；与雌激素处理组相比，雌激素及受体ERα拮抗剂MPP共处理组MTTPL基因表达水平显著降低（P<0.05）；与MPP和雌激素共处理组相比，ERα和ERβ受体拮抗剂ICI和TAM处理组MTTPL基因表达水平无显著变化。综上所述，鸡MTTPL位于内质网中，具有与人MTTP相似的功能结构域；MTTPL和MTTP均在肝中相对高表达，且MTTPL在产蛋期鸡肝的表达显著高于产蛋前期，而MTTP无显著变化。雌激素可通过与ERα受体结合调控鸡MTTPL的表达，而MTTP不受雌激素调控。表明MTTPL可能在鸡产蛋期肝脂质代谢中发挥重要作用。

关键词: 鸡, MTTP, MTTPL, 脂质代谢, 雌激素, 脂蛋白

Abstract: The purpose of the study was to explore the biological characteristics and expression regulation of chicken microsomal triglyceride transfer protein like gene (MTTPL), and lay a foundation for further studying its biological function in lipid metabolism of chicken liver. In this study, firstly, PCR and sequencing were used to clone MTTPL cDNA sequence. Online softwares were used to analyze the structural domain, three-dimensional structure of MTTPL and construct phylogenetic tree. Overexpression vector pcDNA3.1-MTTPL-EGFP was constructed, and co-transfected chicken hepatocellular carcinoma (LMH) with DsRed-λ vector, which expressed endoplasmic reticulum marker protein, for MTTPL subcellular localization. Then tissue samples were collected from Lushi blue-shelled chickens at different weeks of age (8 chickens for 1 day old, 1 week old, 10 week old, 30 week old, respectively). The spatial and temporal expression profiles of chicken MTTPL gene and microsomal triglyceride transporter (MTTP) gene were analyzed by real-time quantitative PCR (qPCR). Then, the effects of estrogen on the expression of MTTPL and MTTP in chicken liver were estimated, twenty hy-line brown chickens in each group were treated with 17β-estradiol at 0.5, 1 and 2 mg·kg^-1 body weight for 12 and 24 h, respectively. Lastly, the regulation mechanism of estrogen on the expression of MTTPL and MTTP were studied, 1, 50 and 100 nmol·L^-1 17β-estradiol and estrogen receptor antagonist were used to treat chicken embryo hepatocytes, respectively. The results showed that chicken MTTPL CDS region was 2 646 bp in length, encoding 881 amino acids, and sharing a common ancestor with human and chicken MTTP. MTTPL was located in endoplasmic reticulum. Chicken MTTPL and MTTP had the same functional domains as human MTTP, and the similarity deviation of three-dimensional structure with human MTTP were 0.090 and 0.064, respectively. MTTPL was highly expressed in liver and kidney tissues of chicken. MTTP was highly expressed in chicken liver, kidney and small intestine. With the growth development of chickens, the expression levels of ApoB and MTTPL in liver were significantly up-regulated (P<0.05), while the expression levels of MTTP in liver was significantly increased before 10-week-old (P<0.05), but no significant change between 10-week-old and 30-week-old chickens (P>0.05). After estrogen treatment for 12 and 24 h, the expression levels of MTTPL and ApoB in chicken liver were significantly up-regulated (P<0.05). MTTP was significantly down-regulated in high concentration group (P<0.05), while no significant change in low concentration group. Compared with the control, estrogen could significantly up-regulate the expression level of ApoB and MTTPL in chicken embryo hepatocytes (P<0.05), while MTTP was not significantly changed. Compared with estrogen treatment group, the expression level of MTTPL gene was significantly decreased in estrogen and ERα antagonist MPP co-treated group (P<0.05). Compared with MPP and ERα antagonist co-treated group, no significant change of MTTPL expression level in ERα and ERβ receptor antagonist ICI or TAM and estrogen co-treated group was observed. In conclusion, chicken MTTPL was located in endoplasmic reticulum, and had the same functional domains as human MTTP. Both MTTPL and MTTP were highly expressed in chicken liver. Compared with pre-laying stage, MTTPL expression in liver of chicken at laying stage was significantly up-regulated, while no significant difference was observed in MTTP expression. Estrogen could regulate the expression of MTTPL via binding with ERα receptor, but MTTP was not affected. It was showed that MTTPL may play an important role in liver lipid metabolism of chicken at the egg-laying stage.

Key words: chicken, MTTP, MTTPL, lipid metabolism, estrogen, lipoprotein

中图分类号:

S831.2

徐春林, 曹玉珠, 夏天, 贾其辉, 王丹丹, 郑航, 田亚东, 康相涛, 蒋瑞瑞, 刘小军, 李红. 鸡微粒体甘油三酯转运蛋白样基因生物学特性及表达调控[J]. 畜牧兽医学报, 2022, 53(9): 2900-2911.

XU Chunlin, CAO Yuzhu, XIA Tian, JIA Qihui, WANG Dandan, ZHENG Hang, TIAN Yadong, KANG Xiangtao, JIANG Ruirui, LIU Xiaojun, LI Hong. Biological Characteristics and Expression Regulation of Chicken Microsomal Triglyceride Transfer Protein Like Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2900-2911.

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鸡微粒体甘油三酯转运蛋白样基因生物学特性及表达调控

Biological Characteristics and Expression Regulation of Chicken Microsomal Triglyceride Transfer Protein Like Gene

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