目的 获得粉尘螨长链脂肪酸转运蛋白（long-chain fatty acid transport protein，FATP）编码基因及其分子特征。方法 根据粉尘螨转录组基因测序结果，获得FATP编码序列片段，据此设计引物，用反转录-聚合酶链式反应从粉尘螨总RNA中扩增全长基因片段，构建原核表达质粒pET28a（+）-FATP。成功获取FATP序列后，利用Expasy、SingaIP 5.0、GOR4和TMpred等软件进行氨基酸序列结构和功能分析，最后利用Clustal Omega及MEGA-X软件进行同源性分析。结果 基因编码序列全长1 071 bp，编码的蛋白质由356个氨基酸组成，其亲水性指数为-0.39，此蛋白为可溶性蛋白，结构稳定，二级结构包括α-螺旋（25.56%）、延伸主链（23.04%）和无规则卷曲（51.40%）。该蛋白存在两处较明显的跨膜区段，由外向内的跨膜区位于1～20位氨基酸，由内向外的跨膜区在100～116位氨基酸。同源氨基酸序列构建的分子进化树中发现与家蚕聚成一簇。结论 获得了粉尘螨FATP编码基因全长及其分子特征，为探讨粉尘螨生理现象、开发粉尘螨控制措施奠定基础。
Objective To identify the gene encoding the long-chain fatty acid transport protein (FATP) of Dermatophagoides farinae and its molecular characteristics. Methods According to the transcriptome sequencing data of D. farinae, we obtained the FATP-coding sequence for designing primers. Then RT-PCR was used to amplify the full-length gene fragment from total RNA of D. farinae, followed by construction of the prokaryotic expression plasmid pET28a(+)-FATP. After obtaining FATP sequence successfully, the structure and function of amino acid sequence were analyzed by using the software of Expasy、singaIP5.0、GOR4 and TMpred, and performed homology analysis through Clustal Omega and MEGA-X. Results The full length of the gene coding sequence was 1 071 bp. The encoded protein was composed of 356 amino acids. It was soluble, with a grand average of hydropathicity of -0.39, and had a stable structure. The secondary structure included alpha-helix (25.56%), extended strand (23.04%), and random coil (51.40%). The protein had two transmembrane regions, The transmembrane area from the outside to the inside is 1-20 aa, and the transmembrane area from the inside to the outside is 100-116 aa. The evolutionary tree derived from homologous amino acid sequences showed that the sequence of D. farinae was clustered with that of silkworm. Conclusion The full length of the FATP-coding gene and the molecular characteristics of FATP are identified in D. farinae, laying a foundation for exploring its physiological phenomena and developing control measures.
[1] Chan TF,Ji KM,Yim AKY,et al. The draft genome,transcriptome,and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens[J]. J Allergy Clin Immunol,2015,135(2):539-548. DOI:10.1016/j.jaci.2014. 09.031.
[2] Patel S, Meher BR. A review on emerging frontiers of house dust mite and cockroach allergy research[J]. Allergol Immunopathol (Madr). 2016,44(6):580-593. DOI:10.1016/j.aller.2015.11. 001.
[3] Thomas WR,Hales BJ,Smith WA. House dust mite allergens in asthma and allergy[J]. Trends Mol Med,2010,16(7):321-328. DOI:10.1016/j.molmed.2010.04.008.
[4] Banerjee S,Resch Y,Chen KW,et al. Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis[J]. J Invest Dermatol,2015,135(1):102-109. DOI:10.1038/jid.2014.271.
[5] Moingeon P. Progress in the development of specific immunotherapies for house dust mite allergies[J]. Exp Rev Vacc,2014,13(12):1463-1473. DOI:10.1586/14760584. 2014.948861.
[6] Li J,Sun B,Huang Y,et al. A multicentre study assessing the prevalence of sensitizations in patients with asthma and/or rhinitis in China[J]. Allergy,2009,64(7):1083-1092. DOI:10.1111/j.1398-9995.2009.01967.
[7] Anderson CM,Stahl A. SLC27 fatty acid transport proteins[J]. Mol Aspects Med,2013,34(2/3):516-528. DOI:10.1016/j.mam.2012.07.010.
[8] Kazantzis M,Stahl A. Fatty acid transport proteins,implications in physiology and disease[J]. Biochim Biophys Acta,2012,1821(5):852-857. DOI:10.1016/j.bbalip.2011.09.010.
[9] Schaffer JE,Lodish HF. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein[J]. Cell,1994,79(3):427-436. DOI:10.1016/0092-8674(94)90252-6.
[10] Chiu CY,Chang TC,Liu SH,et al. The regulatory effects of fish oil and chitosan on hepatic lipogenic signals in high-fat diet-induced obese rats[J]. J Food Drug Anal,2017,25(4):919-930. DOI:10.1016/j.jfda.2016.11.015.
[11] Rodríguez-Cruz M,González RS,Maldonado J,et al. The effect of gestational age on expression of genes involved in uptake,trafficking and synthesis of fatty acids in the rat placenta[J]. Gene,2016,591(2):403-410. DOI:10.1016/j.gene.2016.06. 034.
[12] SeeBle J,Liebisch G,Schmitz G,et al. Palmitate activation by fatty acid transport protein 4 as a model system for hepatocellular apoptosis and steatosis[J]. Biochim Biophys Acta,2015,1851(5):549-565. DOI:10.1016/j.bbalip.2015.01.004.
[13] Dourlen P,Sujkowski A,Wessells R,et al. Fatty acid transport proteins in disease:new insights from invertebrate models[J]. Prog Lipid Res,2015,60:30-40. DOI:10.1016/j.plipres.2015. 08.001.
[14] Mishima T,Miner JH,Morizane M,et al. The expression and function of fatty acid transport protein-2 and -4 in the murine placenta[J]. PLoS One,2011,6(10):e25865. DOI:10.1371/journal.pone.0025865.
[15] Gallardo D,Amills M,Quintanilla R,et al. Mapping and tissue mRNA expression analysis of the pig solute carrier 27A (SLC27A) multigene family[J]. Gene,2013,515(1):220-223. DOI:10.1016/j.gene.2012.11.029.
[16] Black PN,Ahowesso C,Montefusco D,et al. Fatty acid transport proteins:Targeting FATP2 as a gatekeeper involved in the transport of exogenous fatty acids[J]. Medchemcomm,2016,7(4):612-622. DOI:10.1039/C6MD00043F.
[17] Hu XY,Wang YF,Sheikhahmadi A,et al. Effects of glucocorticoids on lipid metabolism and AMPK in broiler chickens'liver[J]. Comp Biochem Physiol B Biochem Mol Biol,2019,232:23-30. DOI:10.1016/j.cbpb.2019.02.001.
[18] Stremmel W,Ehehalt R,Herrmann T,et al. Role of FATP in parenchymal cell fatty acid uptake[J]. Biochim Biophys Acta,2003,33:81-87. DOI:10.1016/S1569-2558(03)33005-X.
[19] Doege H,Stahl A. Protein-mediated fatty acid uptake:novel insights from in vivo models[J]. Physiology (Bethesda),2006,21(4):259-268. DOI:10.1152/physiol.00014.2006.
