家兔矮小同源盒基因(Shox2)的克隆和表达分析

doi:10.11843/j.issn.0366-6964.2019.01.008

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (1): 61-70.doi: 10.11843/j.issn.0366-6964.2019.01.008

家兔矮小同源盒基因(Shox2)的克隆和表达分析

陈瑞^1,2,3, 朱子凤⁴, 黄坚^1,2, 杨晓农^1,2*

1. 西南民族大学生命科学与技术学院, 成都 610041;
2. 青藏高原动物遗传资源保护与利用教育部和四川省重点实验室, 成都 610041;
3. 成都惠泰生物医药有限公司, 成都 610041;
4. 西南民族大学校医院, 成都 610041

收稿日期:2018-06-22 出版日期:2019-01-23 发布日期:2019-01-23
通讯作者: 杨晓农,主要从事小动物疾病研究,E-mail:yangxn058@163.com
作者简介:陈瑞(1989-),男,安徽涡阳人,硕士,主要从事临床兽医与动物模型研究,E-mail:18010619617@163.com
基金资助:
国家重大支撑项目课题（2014BAD13B03）；西南民族大学研究生学位点建设项目（2014XWD-S0906）

Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus

CHEN Rui^1,2,3, ZHU Zifeng⁴, HUANG Jian^1,2, YANG Xiaonong^1,2*

1. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China;
2. Key Laboratory of Ministry of Education and Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Chengdu 610041, China;
3. Chengdu Huitai Biomedicine CO., LTD., Chengdu 610041, China;
4. Hospital of Southwest Minzu University, Chengdu 610041, China

Received:2018-06-22 Online:2019-01-23 Published:2019-01-23

摘要/Abstract

摘要：

为了研究新西兰白兔矮小同源盒基因（Shox2）序列，预测其结构和功能，以及研究Shox2基因的时空表达特征，本研究克隆了新西兰白兔Shox2基因全长CDS区，分析其编码蛋白的同源性、亲疏水性、二级和三级结构特点、系统进化树等生物信息学特征，并用Western blot法验证融合蛋白TrxA-Shox2。分别在胚胎中期（E20.5 d）、胚胎后期（E28.5 d）、幼龄期（出生后10 d）、成年期（A）4个时间点采集3只动物的肾、大血管、真皮、心、大脑、肝、肺、脾、肌肉、胆囊共10种组织，采用实时荧光定量PCR技术研究Shox2基因的时空表达图谱。结果，成功克隆了新西兰白兔Shox2基因（登录号：KP726285）。Shox2基因CDS区全长666 bp，翻译221个氨基酸。生物信息学分析显示，Shox2为碱性、不稳定蛋白，Shox2蛋白氨基酸二级结构中α-螺旋区域占61.99%，无规则卷曲区域占33.48%，延伸链占4.52%。Western blot验证了融合蛋白TrxA-Shox2的表达。家兔Shox2蛋白氨基酸序列与眼镜猴、猩猩、褐家鼠、人的氨基酸序列同源性较高，说明Shox2蛋白在进化中较保守。Shox2基因mRNA在很多器官都有表达，在E20.5 d的家兔胚胎中，Shox2 mRNA在肾、肌肉和胆囊的表达量高，在E28.5 d时表达量较高的是脾、心，幼龄期时表达量较高的是真皮、肝，成年期时表达量较高的是真皮、脾。本研究成功克隆了新西兰白兔Shox2基因，预测了其结构和功能。Shox2基因mRNA在很多器官都有表达，不同的组织和不同时间特征的表达量变化可能与Shox2功能高度相关，并受到严格的调控。

Abstract:

To clone and characterize short stature homeobox 2 (Shox2) gene in New Zealand rabbit, predict its structure and function, and to investigate its expression pattern, the full-length CDS sequence of Shox2 was cloned, and the biological characteristics of Shox2 protein were analyzed in this study, such as homology, hydrophilicity, secondary and tertiary structure characteristics and phylogenetic tree. Western blot was employed to verify fusion protein TrxA-Shox2. Quantitative real-time RT-PCR (RT-qPCR) was employed to detect the expression patterns of Shox2 in kidney, aorta, heart, dermis, lung, brain, liver, spleen, gallbladder and muscle tissues at 4 different developmental phases(E20.5 d, E28.5 d, 10 d and adult) of 3 animals for each phase. Shox2 gene was successfully cloned in present study (GenBank accession number:KP726285), CDS of Shox2 was composed of 666 bp encoding 221 amino acids. According to the results of bioinformatics analysis, Shox2 belonged to the unstable alkaline protein, 61.99% of which amino acids was alpha helix, 33.48% for random coil, 4.52% for extended strand. The result of Western blot verified the expression of fusion protein TrxA-Shox2. The analysis of homology and phylogenetic tree showed that the amino acid sequence of Oryctolagus cuniculus Shox2 protein had high homology and close genetic distance with tarsier, gorilla, rattus norvegicus and human, which meant that Shox2 gene was conservative during evolutionary process. According to the result of RT-qPCR, Shox2 gene universally expressed in most tissues detected in this study. A relatively higher Shox2 mRNA expression level was detected in kidney, gallbladder and muscle tissues in embryo for E20.5 d, in heart and spleen in embryo for E28.5 d, in dermis and liver for 10 d, and in derminds and spleen for adult. In this study, Shox2 gene was successfully cloned and characterized. Shox2 gene universally expressed in all tissues detected, its expression level was related to the function of Shox2 in different tissues and at different developmental stages, which was strictly regulated.

中图分类号:

S829.12

陈瑞, 朱子凤, 黄坚, 杨晓农. 家兔矮小同源盒基因(Shox2)的克隆和表达分析[J]. 畜牧兽医学报, 2019, 50(1): 61-70.

CHEN Rui, ZHU Zifeng, HUANG Jian, YANG Xiaonong. Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 61-70.

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家兔矮小同源盒基因(Shox2)的克隆和表达分析

Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus

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