剑麻AsLEC基因克隆及生物信息学分析
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摘要
单子叶甘露糖结合植物凝集素基因能够有效抑制具有刺吸式口器的同翅目害虫的生长与繁殖,它被证实在掌叶半夏、小麦、烟草、棉花等单子叶和双子叶植物中均具有较好的抗虫效果,但其在剑麻中的功能尚缺乏深入研究。本研究以剑麻为材料,利用RT-PCR技术成功克隆剑麻lectin基因,命名为AsLEC,并对其进行生物信息学分析。结果表明:该基因CDS序列全长为561 bp,编码186个氨基酸,蛋白质分子量预测为19.97 ku,理论等电点为4.96,为疏水性蛋白;同源氨基酸序列比对结果表明剑麻lectin基因与火烧兰、雪花莲、君子兰与菠萝等植物的单子叶甘露糖结合凝集素基因同源性较高,氨基酸匹配度达到48%以上,系统进化树分析表明AsLEC基因与火烧兰亲缘关系更近;对AsLEC基因进行功能结构域分析表明其具有B-lectin基因家族典型特征,属于B-lectin基因家族成员;功能预测发现其具有一段33个氨基酸残基的信号肽,同时在N端第5～27位置处存在一个跨膜α螺旋,表明AsLEC蛋白为分泌型蛋白质,这与B-lectin蛋白的功能特点相吻合;AsLEC蛋白二级结构包含11个β折叠,3个α螺旋;亚细胞定位预测该基因很有可能定位在细胞膜上。剑麻AsLEC基因的克隆对于研究其在剑麻中的抗虫功能具有重要意义,同时丰富了单子叶植物中植物凝集素的相关研究成果。
The monocotyl mannose-binding plant lectin gene can effectively inhibit the growth and reproduction of Homoptera pests with sucking mouthparts, and it was verified that those genes represent better resistance against insects in monocotyledonous and dicotyledonous plants such as Pinellia ternata, Triticum aestivum L., Nicotiana tabacum L.,Gossypium spp. However, its function in sisal is still lack of in-depth study. In this study, sisal was used as materials,and the sisal lectin gene was successfully cloned by RT-PCR and named as AsLEC. Bioinformatics analysis were conducted. The results showed that the full-length CDS of AsLEC gene was 561 bp, encoding 186 amino acids. The protein molecular weight was predicted to be 19.97 ku, and the theoretical isoelectric point was 5.01, which was a hydrophobic protein. The results of homologous amino acid sequence alignment indicated that sisal lectin gene had high homology with the superfamily of monocotyl mannose-binding lectin gene in Epipactis helleborine, Leucojum vernum, Cliviaminiata, Ananas comosus(Linn.) Merr. and so on. The amino acid matching degree was more than 48%. The phylogenetic tree analysis indicated that AsLEC gene was more closely related to the lectin genes in Epipactis helleborine.Functional domain analysis of AsLEC gene suggested that it had the typical characteristics of the B-lectin gene family and belongs to the B-lectin gene family. A signal peptide of 33 amino acid residues was predicted in AsLEC protein, and there was a transmembrane alpha helix at the position from the 5th amino acid to the 27th amino acid in the N-terminus,which implyed that AsLEC protein was a secreted protein and was consistent with the functional characteristics of B-lectin protein. AsLEC protein secondary structure contained 11β-sheets, 3α-helices. Subcellular localization prediction showed that AsLEC gene was likely positioned on the cell membrane. Thecloning of sisal AsLEC gene was of great significance for studying its anti-insect function in sisal, and enriches the related research of plant lectin in monocotyledon.
The monocotyl mannose-binding plant lectin gene can effectively inhibit the growth and reproduction of Homoptera pests with sucking mouthparts, and it was verified that those genes represent better resistance against insects in monocotyledonous and dicotyledonous plants such as Pinellia ternata, Triticum aestivum L., Nicotiana tabacum L.,Gossypium spp. However, its function in sisal is still lack of in-depth study. In this study, sisal was used as materials,and the sisal lectin gene was successfully cloned by RT-PCR and named as AsLEC. Bioinformatics analysis were conducted. The results showed that the full-length CDS of AsLEC gene was 561 bp, encoding 186 amino acids. The protein molecular weight was predicted to be 19.97 ku, and the theoretical isoelectric point was 5.01, which was a hydrophobic protein. The results of homologous amino acid sequence alignment indicated that sisal lectin gene had high homology with the superfamily of monocotyl mannose-binding lectin gene in Epipactis helleborine, Leucojum vernum, Cliviaminiata, Ananas comosus(Linn.) Merr. and so on. The amino acid matching degree was more than 48%. The phylogenetic tree analysis indicated that AsLEC gene was more closely related to the lectin genes in Epipactis helleborine.Functional domain analysis of AsLEC gene suggested that it had the typical characteristics of the B-lectin gene family and belongs to the B-lectin gene family. A signal peptide of 33 amino acid residues was predicted in AsLEC protein, and there was a transmembrane alpha helix at the position from the 5th amino acid to the 27th amino acid in the N-terminus,which implyed that AsLEC protein was a secreted protein and was consistent with the functional characteristics of B-lectin protein. AsLEC protein secondary structure contained 11β-sheets, 3α-helices. Subcellular localization prediction showed that AsLEC gene was likely positioned on the cell membrane. Thecloning of sisal AsLEC gene was of great significance for studying its anti-insect function in sisal, and enriches the related research of plant lectin in monocotyledon.
引文
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[19]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.
[2]马琛,徐涛.植物凝集素抗虫性研究[J].现代园艺,2017(5):31-32.
[3]张晓英,甘敬,尹伟伦.国槐转雪花莲凝集素基因及抗蚜性[J].吉林科学,2010,46(2):51-56.
[4]朱家红,徐靖,畅文军,等.巴西橡胶树HbHEV3基因的克隆和表达分析[J].西北植物学报,2014,34(8):1529-1533.
[5]刘玎,陈劲,刘志,等.掌叶半夏凝集素基因PPA2抗蚜功能分析[J].生物技术通报,2016,32(10):180-187.
[6]徐琼芳,田芳,陈孝.转GNA基因小麦新株系的分子检测和抗蚜虫性鉴定[J].麦类作物学报,2005,25(3):7-10.
[7]Liu S M,Li J,Zhu J Q,et al.Transgenic plants expressing the AalT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests[J].Insect Science,2016,23(2):265-276.
[8]肖松华,刘剑光,吴巧娟.转外源凝集素基因棉花对棉蚜的抗性鉴定[J].棉花学报,2005,17(2):6-10.
[9]肖政,李纪元,范正琪,等.荔波连蕊茶GA2oxl基因的克隆及表达分析[J].林业科学研究,2016,29(1):41-47.
[10]路东晔,贺玉娇,金娜,等.沙柳Sps LAS基因克隆及生物信息学分析[J].分子植物育种,2017,15(2):483-491.
[11]Marchler-Bauer A,Bo Y,Han L Y,et al.CDD/SPARCLE:functional classification of proteins via subfamily domain architectures[J].Nucleic Acids Research,2017,45:D200-D203.
[12]梁辉.转雪花莲凝集素基因小麦抗蚜性及建立高效小麦转化体系的研究[D].武汉:华中农业大学,2005.
[13]Barre A,Bourne Y,Van Damme E J,et al.Mannose-binding plant lectins:Different structural scaffolds for a common sugar-recognition process[J].Biochimie,2001,83(7):645-651.
[14]Hester G,Kaku H,Goldstein I J,Wright C S.Structure of mannose-specific snowdrop(Galanthus nivalis)lectin is representative of a new plant lectin family[J].Nature Structural Biology,1995,2(6):472-479.
[15]王静静.芸豆植物凝集素及其制剂的生殖生物学相关性质研究[D].昆明:云南师范大学,2017.
[16]刘晓娜.植物凝集素基因职务表达载体的构建及遗传转化甘蔗[D].海口:海南大学,2010.
[17]赵欢,彭正松.半夏属植物凝集素的研究进展[J].天然产物研究与开发,2014,26(9):1531-1537.
[18]段晓亮,许兰杰,侯起岭,等.转人工合成中国水仙凝集素基因(s NTL)小麦的获得及其抗麦长管蚜效果分析[J].华北农学报,2016,31(1):8-14.
[19]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.