Amplification and Expression of a Lectin Gene from Agaricus bisporus AS2796
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摘要:
目的 获得双孢蘑菇凝集素基因,了解双孢蘑菇凝集素家族蛋白的功能。 方法 根据已报道的双孢蘑菇凝集素基因序列设计引物,以总DNA为模板扩增双孢蘑菇凝集素基因。将长432 bp的基因亚型克隆后连接至携带His标签的原核表达载体pET-28a,转化大肠杆菌BL21(DE3),经IPTG诱导表达,表达产物经亲和层析纯化后,进行Western blot印迹分析。 结果 扩增的凝集素基因大小为432 bp,重组表达质粒pET-Lectin构建正确,重组凝集素蛋白在大肠杆菌BL21(DE3)中的表达产物约为18 kDa,重组蛋白纯化后纯度达95%以上。 结论 本研究首次扩增双孢蘑菇凝集素基因,并获得纯度较高的双孢蘑菇凝集素蛋白,可利用凝集试验检测其生物学活性,为目的基因的深入研究提供参考依据。 Abstract:Objective A lectin gene from Agaricus bisporus AS2796 was isolated to study the functions of the agglutinin protein in mushrooms. Method Based upon the reported sequences, a primer was designed to amplify the lectin gene with the total DNA as templates. A 432 bp PCR product was purified and connected to the prokaryotic expression vector pET-28a carrying the His label, then transformed into E. coli BL21 (DE3). The IPTG-induced expression product was subsequently purified by affinity chromatography and identified by Western blot. Result The amplified lectin gene was 432 bp with correctly constructed recombinant expression plasmid, pET-lectin. The recombinant agglutinin protein was about 18 kDa and more than 95% pure after purification, Conclusion For the first time, a lectin gene was successfully isolated from A. bisporus and amplified. The agglutinin protein obtained could be used to further study its bioactivity and biosynthesis. -
Key words:
- Agaricus bisporus /
- lectin /
- vector construction /
- prokaryotic expression
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图 4 凝集素融合基因表达产物的SDS-聚丙烯酰胺电泳分析
注:M:低分子量标准蛋白;1:含诱导空载体pET-28a的菌体总蛋白;2:含未诱导空载体pET-28α的菌体总蛋白;3~4:含未诱导重组载体pET-lectin的菌体总蛋白;5~8:含诱导重组载体pET-lectin的菌体总蛋白。
Figure 4. SDS-PAGE on expression product of lectin fusion gene
Note: M: Protein Marker; 1: expression products of induced pET-28α; 2: expression products of pET-28α without induction; 3 and 4: expression products of recombinant pET-lectin without induction; 5–8: expression products of induced recombinant carrier pET-lectin.
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[1] 孙册, 朱政,莫汉庆. 凝集素[M]. 北京: 科学出版社, 1986. [2] SHARON N. History of lectins: from hemagglutinins to biological recognition molecules [J]. Glycobiology, 2004, 14(11): 53R−62R. doi: 10.1093/glycob/cwh122 [3] 吴恩奇, 图力古尔. 蘑菇凝集素及其研究进展 [J]. 菌物研究, 2006, 4(4):69−76. doi: 10.3969/j.issn.1672-3538.2006.04.014WU E Q, BAU TOLGOR. A review of lectins from mushrooms [J]. Journal of Fungal Research, 2006, 4(4): 69−76.(in Chinese) doi: 10.3969/j.issn.1672-3538.2006.04.014 [4] 林玉满, 苏爱华. 长裙竹荪凝集素的分离纯化与部分生化性质 [J]. 中国生物化学与分子生物学报, 2003, 19(2):261−263. doi: 10.3969/j.issn.1007-7626.2003.02.023LIN Y M, SU A H. Isolation, purification and characterization of lectin from Dictyophora indusiata fisch [J]. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(2): 261−263.(in Chinese) doi: 10.3969/j.issn.1007-7626.2003.02.023 [5] 王贺祥, 黄荣春, 吴子斌, 等. 蕈菌凝集素 [J]. 中国食用菌, 1997, 16(1):3−4.WANG H X, HUANG R C, WU Z B, et al. Mushroom lectins [J]. Edible Fungi of China, 1997, 16(1): 3−4.(in Chinese) [6] WANG H X, NG T B, OOI V E C. Lectins from mushrooms [J]. Mycological Research, 1998, 102(8): 897−906. doi: 10.1017/S0953756298006200 [7] 李燕娥, 朱祯, 上官小霞, 等. 棉花转凝集素基因的研究进展 [J]. 中国棉花, 2004, 31(6):5−6. doi: 10.3969/j.issn.1000-632X.2004.06.002LI Y E, ZHU Z, SHANGGUAN X X, et al. Advances in the study of cotton lectin gene [J]. China Cotton, 2004, 31(6): 5−6.(in Chinese) doi: 10.3969/j.issn.1000-632X.2004.06.002 [8] 孙慧, 赵辰光, 仝鑫, 等. 杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能 [J]. 中国生物化学与分子生物学报, 2003, 19(2):210−214. doi: 10.3969/j.issn.1007-7626.2003.02.014SUN H, ZHAO C G, TONG X, et al. Agrocybe aegerita lectin AAVP with antivirus and mycelium differentiation promotion activities [J]. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(2): 210−214.(in Chinese) doi: 10.3969/j.issn.1007-7626.2003.02.014 [9] MOKOCHINSKI J B, LÓPEZ B G C, SOVRANI V, et al. Production of Agaricus brasiliensis mycelium from food industry residues as a source of antioxidants and essential fatty acids [J]. International Journal of Food Science & Technology, 2015, 50(9): 2052−2058. [10] KINO K, MIZUMOTO K, SONE T, et al. An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice [J]. Diabetologia, 1990, 33(12): 713−718. doi: 10.1007/BF00400340 [11] PEUMANS W J, BARRE A, HAO Q, et al. Higher plants developed structurally different motifs to recognize foreign glycans [J]. Trends in Glycoscience and Glycotechnology, 2000, 12(64): 83−101. doi: 10.4052/tigg.12.83 [12] SUN H, ZHAO C G, TONG X, et al. A lectin with mycelia differentiation and antiphytovirus activities from the edible mushroom Agrocybe aegerita [J]. BMB Reports, 2003, 36(2): 214−222. doi: 10.5483/BMBRep.2003.36.2.214 [13] 赵秀云. 天南星凝集素和石蒜凝集素基因的克隆及抗虫研究 [D]. 上海: 复旦大学, 2003.ZHAO X Y. Cloning of lectin genes from Arisaema hoterophyllum and Lycoris radiate and the insect resistance research of their protein production [D]. Shanghai: Fudan University, 2003.(in Chinese) [14] NGAI P H K, NG T. A mushroom (Ganoderma capense) lectin with spectacular thermostability, potent mitogenic activity on splenocytes, and antiproliferative activity toward tumor cells [J]. Biochemical and Biophysical Research Communications, 2004, 314(4): 988−993. doi: 10.1016/j.bbrc.2003.12.196 [15] XIANG Y, SONG M, WEI Z Y, et al. A jacalin-related lectin-like gene in wheat is a component of the plant defence system [J]. Journal of Experimental Botany, 2011, 62(15): 5471−5483. doi: 10.1093/jxb/err226 [16] 周英, 谢红卫, 刘长爱, 等. 豆科凝集素基因Le4的克隆及其表达产物对蚜虫的抗性 [J]. 基因组学与应用生物学, 2016, 35(12):3474−3480.ZHOU Y, XIE H W, LIU C A, et al. Cloning of lectin Le4 gene from leguminosae and the aphid resistance effect of its protein production [J]. Genomics and Applied Biology, 2016, 35(12): 3474−3480.(in Chinese) [17] FURUKAWA K, YING R, NAKAJIMA T, et al. Hemagglutinins in fungus extracts and their blood group specificity [J]. Experimental and Clinical Immunogenetics, 1995, 12(4): 223−231. [18] 刘艳如, 余萍, 郑怡. 12种食用菌凝集素的筛选 [J]. 福建师范大学学报(自然科学版), 2003, 19(2):65−68.LIU Y R, YU P, ZHENG Y. Screening of lectins from twelve varieties of edible mushroom [J]. Journal of Fujian Normal University(Natural Science Edition), 2003, 19(2): 65−68.(in Chinese) [19] 张迪, 林勇, 杨晓钦, 等. 双孢蘑菇2796凝集素的提取及其部分理化性质研究 [J]. 福建农业学报, 2011, 26(3):457−461. doi: 10.3969/j.issn.1008-0384.2011.03.024ZHANG D, LIN Y, YANG X Q, et al. Extraction and partial characterization of a lectin from Agaricus bisporus strain AS2796 [J]. Fujian Journal of Agricultural Sciences, 2011, 26(3): 457−461.(in Chinese) doi: 10.3969/j.issn.1008-0384.2011.03.024 [20] 陈卓. 拟南芥类凝集素基因AtLECLl的功能研究 [D]. 石家庄 河北农业大学, 2013.CHEN Z. Functional analysis of putative lectin gene AtLECLl in Arabidopsis thaliana [D].. Shijiazhuang: Agricultural University of Hebei, 2013. (in Chinese) [21] 郑璐平, 谢荔岩, 姚锦爱, 等. 孔石莼凝集素蛋白基因的克隆与表达 [J]. 激光生物学报, 2008, 17(6):762−767. doi: 10.3969/j.issn.1007-7146.2008.06.011ZHENG L P, XIE L Y, YAO J A, et al. Cloning and expression of lectin gene of Ulva pertusa [J]. Acta Laser Biology Sinica, 2008, 17(6): 762−767.(in Chinese) doi: 10.3969/j.issn.1007-7146.2008.06.011 [22] 孙素荣, 张智, 李素丽, 等. 新疆黄精凝集素基因的克隆、序列分析和表达 [J]. 生物工程学报, 2008, 24(3):387−394. doi: 10.3321/j.issn:1000-3061.2008.03.008SUN S R, ZHANG Z, LI S L, et al. Cloning, sequencing analysis and expression of a putative mannose-binding lectin gene from Polygonatum roseum in Xinjiang [J]. Chinese Journal of Biotechnology, 2008, 24(3): 387−394.(in Chinese) doi: 10.3321/j.issn:1000-3061.2008.03.008 [23] 郭旭. 两种新型家蚕凝集素基因的克隆及表达 [D]. 武汉: 华中师范大学, 2010.GUO X. cDNA cloning and expression analysis of two novel C-type lectins from the silkworm [D]. Wuhan: Central China Normal University, 2010.(in Chinese) [24] 方标, 王丕武, 于雷, 等. 大豆凝集素基因Le2克隆及转化 [J]. 大豆科学, 2011, 30(4):706−709.FANG B, WANG P W, YU L, et al. Cloning and transformation of soybean lectin gene Le2 into tobacco [J]. Soybean Science, 2011, 30(4): 706−709.(in Chinese) [25] 孙科源, 纠敏, 张新春, 等. 无核荔枝凝集素基因的克隆与表达分析 [J]. 热带作物学报, 2011, 32(7):1309−1313. doi: 10.3969/j.issn.1000-2561.2011.07.024SUN K Y, JIU M, ZHANG X C, et al. Cloning and expression analysis of a lectin gene from Wuhe Litchi [J]. Chinese Journal of Tropical Crops, 2011, 32(7): 1309−1313.(in Chinese) doi: 10.3969/j.issn.1000-2561.2011.07.024 [26] 陈仁良, 王威, 严俊杰, 等. 金针菇凝集素基因Fv-mJRL1的生物信息学分析及其共表达 [J]. 厦门大学学报(自然科学版), 2016, 55(3):323−329.CHEN R L, WANG W, YAN J J, et al. Bioinformatic analysis and co-expression study of jacalin-related lectin gene Fv-mJRL1 in Flammulina velutipes [J]. Journal of Xiamen University(Natural Science Edition), 2016, 55(3): 323−329.(in Chinese) [27] 吕明珠. 柞蚕凝集素基因ApCTL的克隆及表达分析 [D]. 大连: 大连理工大学, 2018.LÜ M Z. Cloning and expression analysis of ApCTL gene from Antheraea pernyi [D]. Dalian: Dalian University of Technology, 2018.(in Chinese) [28] 王洪乐, 齐连芬, 杨超沙, 等. 掌叶半夏凝集素基因克隆及原核表达 [J]. 华北农学报, 2018, 33(1):109−114. doi: 10.7668/hbnxb.2018.01.017WANG H L, QI L F, YANG C S, et al. The agglutinin gene cloning of Pinellia pedatisecta and prokaryotic expression [J]. Acta Agriculturae Boreali-Sinica, 2018, 33(1): 109−114.(in Chinese) doi: 10.7668/hbnxb.2018.01.017 [29] 彭博, 李炳娟, 关文强, 等. 双孢蘑菇转录组测序及褐变相关基因的挖掘 [J]. 食品科学, 2019, 40(2):126−132. doi: 10.7506/spkx1002-6630-20180510-154PENG B, LI B J, GUAN W Q, et al. Transcriptome sequencing of Agaricus bisporus and mining of genes involved in browning [J]. Food Science, 2019, 40(2): 126−132.(in Chinese) doi: 10.7506/spkx1002-6630-20180510-154 [30] 李河, 周国英, 刘君昂. 双孢蘑菇褐腐病病原菌的分离及分子鉴定 [J]. 食用菌学报, 2009, 16(2):74−76. doi: 10.3969/j.issn.1005-9873.2009.02.014LI H, ZHOU G Y, LIU J A. Isolation and molecular identification of a pathogen causing brown rot in Agaricus bisporus [J]. Acta Edulis Fungi, 2009, 16(2): 74−76.(in Chinese) doi: 10.3969/j.issn.1005-9873.2009.02.014 [31] HILDER V A, POWELL K S, GATEHOUSE A M R, et al. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids [J]. Transgenic Research, 1995, 4(1): 18−25. doi: 10.1007/BF01976497 [32] POWELL K S, GATEHOUSE A M R, HILDER V A, et al. Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps [J]. Entomologia Experimentalis et Applicata, 1993, 66(2): 119−126. doi: 10.1111/j.1570-7458.1993.tb00699.x [33] TANG K, ZHAO E, SUN X, et al. Production of transgenic rice homozygous lines with enhanced resistance to the rice brown planthopper [J]. Acta Biotechnologica, 2001, 21(2): 117−128. doi: 10.1002/1521-3846(200105)21:2<117::AID-ABIO117>3.0.CO;2-Y