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烟草碱性β-1,3-葡聚糖酶具有较强的抗真菌病害等作用。本研究根据烟草Samsun与抗真菌作用有关的碱性β-1,3-葡聚糖酶基因序列设计引物,以烟草(Nicotiana tabacum)品种Xanthi NN基因组DNA为模板,进行PCR扩增,后将扩增产物与pUC57-T载体重组,再以该重组体转化大肠杆菌DH5α,在对阳性克隆进行鉴定后测序,并对其编码酶蛋白进行了一级、二级、三级结构及蛋白质同源性比较等生物信息学分析。研究结果表明,该基因全长1 868 bp,包含2个外显子和1个内含子;除终止密码子外,共编码359个氨基酸(GenBank登录号:EU867448)。该酶分子量为28.4 kDa,pI为9.72,是一个碱性蛋白;具有多个螺旋、转角、延伸链和无规卷曲等二级结构,其中α-螺旋占35.65%,β-转角占5.57%,延伸链占19.22%,无规卷曲占39.55%;三级结构同源建模预测显示,它与香蕉(Musa supientum)β-1,3-葡聚糖酶(PDB number 2cygA)具有60%的一致性;推测该酶蛋白与颠茄(Atropa belladonna)、马铃薯(Solanum tuberosum)等茄科植物的β-1,3-葡聚糖酶编码序列同源性分别达到89%和81%。本研究结果为进一步开展烟草抗真菌的β-1,3-葡聚糖酶的基因工程研究奠定了重要基础。
Tobacco alkaline β-1, 3-glucanase has strong antifungal and other effects. In this study, primers were designed based on the sequence of tobacco β-1,3-glucanase related to antifungal activity of Samsun. The genomic DNA of Nicotiana tabacum Xanthi NN was used as template to amplify The amplified product was recombined with pUC57-T vector, and then the recombinant was transformed into E. coli DH5α. The positive clones were identified and sequenced. The primary, secondary, tertiary structure and protein homology Comparative bioinformatics analysis. The results showed that the gene was 1 868 bp in length and contained two exons and one intron. The gene contained 359 amino acids (GenBank accession number: EU867448) except for the stop codon. The enzyme has a molecular weight of 28.4 kDa and a pI of 9.72, which is a basic protein. It has secondary structures such as helix, angle, extension chain and random coil. The α-helix accounted for 35.65% and β-turn accounted for 5.57% The extension chain accounted for 19.22% and the random coil accounted for 39.55%. The prediction of tertiary structure homology modeling showed that it was 60% identical to that of Musa supientum PDB number 2cygA It is speculated that the homology of β-1,3-glucanase of this enzyme protein to Solanaceae such as Atropa belladonna and Solanum tuberosum is 89% and 81% respectively. The results of this study laid an important foundation for further genetic engineering of tobacco antifungal β-1,3-glucanase.