Cloning and Analysis of Tree Shrew Mfsd2a Gene and Detection of Its Expression in Different Tissues

doi:10.3969/j.issn.1674-5817.2019.03.002

Abstract

Abstract: Objective To clone and sequence the major facilitator superfamily domain containing 2a(Mfsd2a) gene of tree shrew and analyze its bioinformatics characteristics, and quantitatively detect its expression in different tissues. Methods Total RNA was extracted from the brain and spinal cord of tree shrews, Mfsd2a gene was amplified by RT-PCR. Bioinformatics software was used to analyze its sequence characteristics, system evolution, phylogenetics, structure and physicochemical properties of the encoding proteins. The expression of Mfsd2a gene in different tissues were detected by qPCR using β-actin as an internal reference. Results The full-length of Mfsd2a gene fragment was 1 796 bp, which was highly consistent with the tree shrew Mfsd2a gene sequence published on NCBI. The coding sequence was 1 464 bp, which encoded 488 amino acids. It was also homologous to the MFS transporter superfamily. The tree shrew Mfsd2a protein had a distinct hydrophobic region, but no signal peptide, and its secondary structural elements were consist of alpha helix, beta sheet, random coil and extended strand. Twelve transmembrane structures were found by OCTOPUS analysis. Two N-glycosylation sites were predicted by modified structure. Subcellular localization revealed that it may be mainly distributed in cell membrane and endoplasmic reticulum. qPCR results showed that Mfsd2a were expressed in different tissues of tree shrew, and the it was relatively high in brain and spinal cord. Conclusion The Mfsd2a gene was successfully cloned, and a quantitative detection method for the expression of Mfsd2a was established, which might provide theoretical and technical support for further study of Mfsd2a gene function using tree shrew neurological disease model in the future.

Key words: Tree shrew, Major facilitator superfamily domain containing 2a (Mfsd2a), Clone, Analysis, Expression

CLC Number:

Q95-33

WANG Wen-guang, KUANG De-xuan, LI Na, LU Cai-xia, HAN Yuan-yuan, TONG Pin-fen, SUN Xiao-mei, DAI Jie-jie. Cloning and Analysis of Tree Shrew Mfsd2a Gene and Detection of Its Expression in Different Tissues[J]. Laboratory Animal and Comparative Medicine, 2019, 39(3): 178-186.

References

[1] Ethayathulla AS, Yousef MS, Amin A, et al.Structure-based mechanism for Na(+)/melibiose symport by MelB[J]. Nat Commun, 2014, 5:3009.
[2] Nguyen LN, Ma D, Shui G, et al.Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid[J]. Nature, 2014, 509(7501):503-506.
[3] Ben-Zvi A, Lacoste B, Kur E, et al.Mfsd2a is critical for the formation and function of the blood-brain barrier[J]. Nature, 2014, 509(7501):507-511.
[4] Berger JH, Charron MJ, Silver DL, et al.Major facilitator superfamily domain-containing protein 2a (MFSD2A) has roles in body growth, motor function, and lipid metabolism[J]. PLoS One, 2012, 7(11):e50629.
[5] Oppelt C, Wutzler R, von Holst D. Characterisation of MHC class II DRB genes in the northern tree shrew (Tupaia belangeri)[J]. Immunogenetics, 2010, 62:613-622.
[6] Zhang L, Zhang H, Zhu W, et al.Energy metabolism, thermogenesis and body mass regulation in tree shrew (Tupaia belangeri) during subsequent cold and warm acclimation[J]. Comp Biochem Physiol: A Mol Integr Physiol, 2012, 162:437-442.
[7] Ping S, Wang F, Zhang Y, et al.Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)[J]. Theriogenology, 2011, 76: 39-46.
[8] Brack M.Malignant Leydig cell tumour in a Tupaia belangeri: case report and literature review of male genital tumours in non-human primates[J]. Lab Anim, 1988, 22:131-134.
[9] 周顺长, 蔡红琳. 树鼩中枢神经系统的某些解剖学特点[J]. 上海实验动物科学, 1993(3):176-177.
[10] 唐代彬, 李树清. 树鼩脑缺血时海马微环境与血脑屏障通透性改变的可能机制[J]. 中国微循环, 2008, 12:76-80,64.
[11] 张颖, 李树清, 李凡. 树鼩海马微灌流谷氨酸和钙所致线粒体应激及银杏内酯B的影响[J]. 中国微循环, 2007, 11:158-162, 219.
[12] Ma KL, Gao JH, Huang ZQ, et al.Motor Function in MPTP-Treated Tree Shrews (Tupaia belangeri chinensis)[J]. Neurochemical Research, 2013, 38(9):1935-1940.
[13] 欧阳轶强, 胡冰, 张莹, 等. 阿尔茨海默病树鼩模型建立的探索[J].四川动物, 2016, 35(2):270-274.
[14] 吴元桢, 欧阳轶强, 黄玲, 等. 新生树鼩海马神经干细胞的体外培养与鉴定[J]. 广西医学, 2012, 34:257-260.
[15] 李娟, 曾晓锋, 杨根梦, 等. 新生树鼩多巴胺神经元的体外培养[J]. 中国比较医学杂志, 2018, 28(7):82-86.
[16] 李纯, 孙春玉, 陈静, 等. 主要协同转运蛋白超家族的研究进展[J]. 生物技术通报, 2018, 34(8):43-49.
[17] Esnault C, Priet S, Ribet D, et al.A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived,human syncytin-2[J]. Proc Natl Acad Sci U S A, 2008, 105(45):17532-17537.
[18] Yang YR, Xiong XY, Liu J, et al.Mfsd2a (major facilitator superfamily domain containing 2a) attenuates intracerebral hemorrhage-induced blood-brain barrier disruption by inhibiting vesicular transcytosis[J]. J Am Heart Assoc, 2017, 6(7):e005811.