猪FUT2基因密码子使用偏好性分析及优化

doi:10.16431/j.cnki.1671-7236.2018.07.017

摘要/Abstract

摘要：

为了揭示猪α-（1，2）岩藻糖转移酶2（FUT2）基因密码子使用特性并提高其在外源宿主内的表达量，本研究综合运用EMBOSS Explorer网站和CodonW软件包分析了猪FUT2基因的密码子使用偏好性的相关参数，并与3种模式生物（大肠杆菌、酵母菌和果蝇）基因组密码子使用模式进行比较，最后参照与之最为相近的模式生物基因组密码子使用方式，利用JCat和OPTIMIZER两个密码子优化网站对猪FUT2基因密码子进行优化。结果显示，猪FUT2基因表达水平不高，存在24种偏好密码子，且这24种偏好密码子中有23种以G/C结尾；猪FUT2基因与果蝇基因组密码子使用模式的差异小于大肠杆菌和酵母基因组，表明果蝇更适合猪FUT2基因的外源表达；根据果蝇基因组密码子使用模式优化猪FUT2基因密码子，优化后其适应指数有了明显提高，而整体GC含量没有明显变化，说明在理论上猪FUT2基因优化成功。本研究从翻译水平上揭示了猪FUT2基因的密码子使用特性，为其在遗传改良中选择最佳外源表达系统及提高其在宿主细胞内的表达水平提供一定的理论依据。

关键词: 猪; FUT2基因; 密码子使用偏好性; 密码子优化

Abstract:

To reveal the codon usage pattern of porcine α-(1,2) fucosyltransferase 2 (FUT2) gene and increase its expression level in exogenous host,codon usage bias parameters of porcine FUT2 gene was analyzed using CodonW and EMBOSS Explorer,and then compared it with Drosophila, yeast and Escherichia coli genomes.Finally,according to the most closely codon usage pattern of model organism,the porcine FUT2 gene codon was optimized using JCat and OPTIMIZER.The results showed the expression level of porcine FUT2 gene was low,and it had 24 biased codons,23 of which biased to end with G/C at the third position.The differences in codon usage frequency between porcine FUT2 gene and Drosophila genome were less than those in Escherichia coli and yeast genomes,which proved Drosophila was more suitable for expressing the porcine FUT2 gene than Escherichia coli and yeast.The Drosophila genome was employed to optimize porcine FUT2 gene.The codon adaptation index of optimized porcine FUT2 gene was significantly improved,while the GC content of its CDS sequence was not obviously changed,indicating that the porcine FUT2 gene was successfully optimized in theory.This research revealed the codon usage pattern of porcine FUT2 gene from the translation level,and provided theoretical basis for selecting the best exogenous expression system and improving the expression level of the porcine FUT2 gene in host cells.

Key words: porcine; FUT2 gene; codon usage bias; codon optimization

中图分类号:

S813.3

戴开宇, 黄焱杰, 吴丽思, 包文斌, 吴圣龙. 猪FUT2基因密码子使用偏好性分析及优化[J]. 《中国畜牧兽医》, 2018, 45(7): 1863-1872.

DAI Kaiyu, HUANG Yanjie, WU Lisi, BAO Wenbin, WU Shenglong. Analysis and Optimization of Codon Usage Bias of Porcine FUT2 Gene[J]. , 2018, 45(7): 1863-1872.

参考文献

[1] QIN W Y,GAN L N,XIA R W,et al.New insights into the codon usage patterns of the bactericidal/permeability-increasing (BPI) gene across nine species[J].Gene,2017,616:45-51.
[2] ZHANG L,JIN L G,LUO L,et al.Analysis of nuclear gene codon bias on soybean genome and transcriptome[J].Acta Agronomica Sinice,2011,37(6):965-974.
[3] ANGELLOTTI M C,BHUIYAN S B,CHEN G,et al.CodonO:Codon usage bias analysis within and across genomes[J].Nucleic Acids Research,2007,35(Suppl_2):W132-W136.
[4] WU A B,CHEN H D,TANG Z Z,et al.Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris[J].Chemico-Biological Interactions,2008,175(1):403-405.
[5] 蒋磊,周明旭,夏芃芃,等.产肠毒素大肠杆菌感染的分子致病机制[J].中国兽医学报,2014,34(9):1551-1560. JIANG L,ZHOU M X,XIA P P,et al.Molecular pathogenic mechanisms of enterotoxigenic Escherichia coli infection[J].Chinese Journal of Veterinary Science,2014,34(9):1551-1560.(in Chinese)
[6] LONARDI E,MOONENS K,BUTS L,et al.Structural sampling of glycan interaction profiles reveals mucosal receptors for fimbrial adhesins of enterotoxigenic Escherichia coli[J].Biology,2013,2(3):894-917.
[7] 钦伟云,甘丽娜,赵呈祥,等.东串猪FUT2基因CDS扩增、序列分析及组织表达谱检测[J].中国畜牧兽医,2017,44(1):1-11. QIN W Y,GAN L N,ZHAO C X,et al.Amplification,sequence analysis and tissue expression detection of FUT2 gene coding regions of Dongchuan pigs[J].China Animal Husbandry & Veterinary Medicine,2017,44(1):1-11.(in Chinese)
[8] HURD E A,HOLMEN J M,HANSSON G C,et al.Gastrointestinal mucins of Fut2-null mice lack terminal fucosylation without affecting colonization by Candida albicans[J].Glycobiology,2005,15(10):1002-1007.
[9] 赵美华,龚陈,楼江明,等.黏蛋白与炎症性肠病关系的研究进展[J].世界华人消化杂志,2014,22(27):4100-4106. ZHAO M H,GONG C,LOU J M,et al.Relationship between mucins and inflammatory bowel disease[J].World Chinese Journal of Digestology,2014,22(27):4100-4106.(in Chinese)
[10] 李平.籽粒苋C₄关键酶基因(NAD-ME、PEPC)的克隆及NAD-ME功能预测[D].太原:山西大学,2010. LI P.Cloning key enzyme genes (NAD-ME,PEPC) of C₄ photosynthesis from Amaranthus hypochondriacus L. and function prediction of NAD-ME[D].Taiyuan:Shanxi University,2010.(in Chinese)
[11] 杨金玲,高丽丽,朱平,等.蝎毒镇痛活性肽基因BmK AngM1的密码子优化及其真核表达分析[J].药学学报,2012,47(10):1389-1393. YANG J L,GAO L L,ZHU P,et al.Codon optimization and eukaryotic expression analysis of the analgesic peptide gene BmK AngM1 from Buthus martensii Karsch[J].Acta Pharmaceutica Sinica,2012,47(10):1389-1393.(in Chinese)
[12] YANG J,LIU L.Codon optimization through a two-step gene synthesis leads to a high-level expression of Aspergillus niger lip2 gene in Pichia pastoris[J].Journal of Molecular Catalysis B:Enzymatic,2010,63(3):164-169.
[13] FEDOROV A,SAXONOV S,GILBERT W.Regularities of context-dependent codon bias in eukaryoticgenes[J].Nucleic Acids Research,2002,30:1192-1197.
[14] ANGOV E.Codon usage:Nature's roadmap to expression and folding of proteins[J].Biotechnology Journal,2011,6(6):650-659.
[15] TULLER T,WALDMAN Y Y,KUPIEC M,et al.Translation efficiency is determined by both codon bias and folding energy[J].Proceedings of the National Academy of Sciences,2010,107(8):3645-3650.
[16] FUGLSANG A.The ‘effective number of codons’ revisited[J].Biochemical and Biophysical Research Communications,2004,317(3):957-964.
[17] WAN X F,XU D,KLEINHOFS A,et al.Quantitative relationship between synonymous codon usage bias and GC composition across unicellular genomes[J].BMC Evolutionary Biology,2004,4(1):19.
[18] BURGESS-BROWN N A,SHARMA S,SOBOTT F,et al.Codon optimization can improve expression of human genes in Escherichia coli:A multi-gene study[J].Protein Expression and Purification,2008,59(1):94-102.
[19] 李玉权,叶远浓,陶小买,等.短短芽孢杆菌GZDF3全基因组密码子偏好性分析[J].基因组学与应用生物学,2017,12:5187-5190. LI Y Q,YE Y N,TAO X M,et al.Analysis of codon bias in whole genome of Brevibacillus brevis GZDF3[J].Genomics and Applied Biology,2017,12:5187-5190.(in Chinese)
[20] ABRANTES J,POSADA D,GUILLON P,et al.Widespread gene conversion of alpha-2-fucosyltransferase genes in mammals[J].Journal of Molecular Evolution,2009,69(1):22.
[21] LINDESMITH L,MOE C,MARIONNEAU S,et al.Human susceptibility and resistance to Norwalk virus infection[J]. Nature Medicine,2003,9(5):548-553.
[22] WHYTE J J,PRATHER R S.Genetic modifications of pigs for medicine and agriculture[J].Molecular Reproduction and Development,2011,78(10-11):879-891.
[23] CODDENS A,DISWALL M,ÅNGSTRÖM J,et al.Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli[J].Journal of Biological Chemistry,2009,284(15):9713-9726.
[24] 崔丹,温洁霞,霍珊珊,等.密码子优化提高猪IL-7在HEK293T细胞表达的研究[J].河北农业大学学报,2017,40(6):88-92. CUI D,WEN J X,HUO S S,et al.Codon optimization enchances porcine interleukin-7 expression in HEK293T cells[J].Journal of Agricultural University of Hebei,2017,40(6):88-92.(in Chinese)