松材线虫Bx-ubc-3基因克隆及泛素通路鉴定

文章编号：1006-8023（2019）05-0009-07

Abstract：The ubiquitin pathway is able to regulate many critical cellular functions. In order to explore the existence of the ubiquitin pathway and a key enzyme gene in ubiquitin pathway， ubiquitin-conjugating enzyme named as ubc3 in B. xylophilus， a cDNA library of B. xylophilus was constructed and sequenced， ORF analysis， homology analysis， phylogenetic tree construction， protein interaction prediction， docking and molecular dynamics simulation were carried out. The full-length cDNA generated by the nematode spliced leader method was 957 bp. Analysis of the Bx-ubc-3 cDNA sequence indicated that its open reading frame （ORF） was 783 bp and 245 amino acids were encoded. Comparison of the identified amino acid sequence with the current databases revealed extensive homology of Bx-ubc-3 to all known ubiquitin-conjugating enzymes. The modeling and prediction of protein interaction， docking and molecular dynamics simulations showed that the binding sites in Bx-ubc-3 were located in α helix， Loop 1 （L1） ring and Loop 2 （L2） ring. The thioester-forming Cys103， which was located in L2， constituted a catalytic cleft. The interfaces of Bx-ubc-3 to Bx-Rbx1 and Bx-ubc-3 to Bx-Ubiquitin were also supported by several obvious hydrogen bond interactions. Results show that there is a theoretic ubiquitin pathway in B. xylophilus， and support the notion that Bx-ubc-3 is a potential target for developing potent antinematodal drugs against B. xylophilus.

Keywords：Bursaphelenchus xylophilus; ubiquitin-conjugating enzyme 3; E2; ubiquitin pathway

0 引言

泛素化是一種重要的转录后修饰方式[1]，控制着真核生物整个蛋白质组。泛素途径是由泛素激活酶（Ubiquitin-activating enzyme， E1）、泛素结合酶（Ubiquitin-conjugating enzyme， E2）和泛素连接酶（Ubiquitin-protein ligase， E3）介导的级联反应[2]，是细胞内蛋白质选择性降解的重要途径。E2是泛素途径的关键酶，它与E1、E3通过蛋白间的相互作用使泛素与靶蛋白结合，形成一条多泛素链，从而使靶蛋白被26S蛋白酶体识别降解[3]。E2家族蛋白都有着相似的催化结构域，这些催化结构域都包含形成E2-泛素硫酯复合物的活性位点半胱氨酸（thioester-forming cysteine）。在对酵母的研究中，ubc-3（CDC34的同源基因）被证明是E2家族成员[4]，编码Ⅲ类E2，负责细胞周期G1期的蛋白降解[5]。尽管已有研究表明E2在酵母等模式生物的细胞中起着重要作用[6]，但是目前国内外对于植物寄生线虫中这类分子的研究仍然非常有限。

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