摘要
为探究松材线虫(Bursaphelenchus xylophilus)耐药的分子机理,对松材线虫耐药基因Bx-mrp1进行鉴定并对其蛋白结构及功能进行了研究。在松材线虫基因组数据中得到松材线虫与秀丽线虫(Caenorhabditis elegans) mrp1具有同源性的基因,命名为Bx-mrp1,并比较了松材线虫Bx-mrp1与相近线虫属之间的mrp基因差异。对松材线虫Bx-mrp1完整蛋白编码区(CDS)进行PCR扩增后,运用RNAi技术对该基因进行基因沉默,分析沉默该基因后对松材线虫死亡率的影响。通过PyM OL软件对Bx-MRP1蛋白质进行三维结构预测。结果表明:在NCBI中,通过BLAST比对,Bx-mrp1与秀丽线虫mrp1同源性为43%。Bx-MRP1蛋白质包含32个α螺旋和18个β折叠,以及含有3个TMD和2个NBD结构,具有多个跨膜结构域,表明Bx-mrp1具有跨膜运输的功能。通过PCR得到松材线虫Bx-mrp1基因CDS区全长为4 659 bp。在对Bx-mrp1基因沉默后,发现RNAi处理组的松材线虫在同质量浓度的苦参碱药剂胁迫下的死亡率有所增加,在多药耐药生理过程中起正向调控作用。
We explored the molecular mechanism of multidrug resistance in Bursaphelenchus xylophilus,and identified the multidrug resistance gene Bx-mrp1 of gene silencing.The homologous gene of the Caenorhabditis elegans mrp1 was identified and named as Bx-mrp1 according the genome data of B.xylophilus.And then compared that with different nematodes,Bx-mrp1 intact protein coding region(CDS)amplification was performed using PCR.Bx-mrp1 silencing was performed using RNAi technique to analyze the effect of silencing the gene on resistance to B.xylophilus.The effect of silence on the mortality of B.xylophilus was analyzed.The third-class structure of Bx-MRP1 was predicted by PyMOL software.The results showed that 43%homology of Bx-mrp1 with C.elegans mrp1 was detected by BLAST in NCBI.The Bx-MRP1 protein contains 32α-helix and 18β-sheets,as well as containing three TMDs and two NBDs,with multiple transmembrane domains.So Bx-mrp1 have the function of transmembrane transport.The total length of Bx-mrp1 CDS was 4 659 bp by PCR.After Bx-mrp1 silencing,the mortality of B.xylophilus of RNAi treated group was increased under the same concentration of matrine agent.Therefore,Bx-mrp1 played a positive role in the multidrug resistance physiological process.
作者
郝昕
王峰
马玲
王博文
马岩
Hao Xin;Wang Feng;Ma Ling;Wang Bowen;Ma Yan(Northeast Forestry University,Harbin 150040,P.R.China;Harbin University of Commerce)
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2018年第9期89-92,97,共5页
Journal of Northeast Forestry University
基金
国家自然科学基金项目(31570637)
黑龙江省自然科学基金项目(ZD201404)
