褐飞虱两个dynamin-1-like基因的克

发布时间：2018-05-09 05:13

  本文选题：褐飞虱 + Dynamin--like　； 参考：《中国水稻科学》2017年04期

【摘要】：【目的】研究dynamin-1-like的两个基因NlDNM1L-1和NlDNM1L-2在褐飞虱中的生物学功能。【方法】通过聚合酶链式反应扩增褐飞虱dynamin-1-like的两个基因,并对其生物信息学进行分析。通过荧光定量PCR技术(qPCR)检测了这两个基因在褐飞虱不同组织和不同发育阶段中的相对表达量。构建原核表达载体,在表达菌株Rosetta中诱导重组目的蛋白的表达。通过Ni柱亲和层析纯化目的蛋白,并将纯化得到的蛋白免疫新西兰大白兔,得到多克隆抗体,并用ELISA检测抗体效价,Western blotting检测抗体特异性。利用上述抗体,通过免疫荧光实验观察目的蛋白在褐飞虱卵巢中的表达和定位。【结果】克隆并鉴定了两个dynamin-1-like基因,分别命名为NlDNM1L-1和NlDNM1L-2(Gen Bank登录号分别为KY082900、KY082901)。系统进化树表明,NlDNM1L-1和NlD NM1L-2在半翅目昆虫中较为保守。蛋白结构预测和基因时空表达分析说明,NlDNM1L-1和NlDNM1L-2在褐飞虱中可能有着不同的功能。ELISA和Western blotting结果表明,制备的NlDNM1L-1和NlDNM1L-2多克隆抗体效价高、特异性好。免疫荧光实验结果显示,NlDNM1L-1和NlDNM1L-2在褐飞虱卵巢滤泡细胞中普遍表达,可能与褐飞虱卵巢发育和成熟有关,它们也可能与类酵母共生菌入侵褐飞虱卵巢有关。【结论】获得了NlDNM1L-1和NlDNM1L-2基因序列,了解了其基本的生物信息,并阐明了其时空表达特征;成功制备其多克隆抗体,并初步了解了NlDNM1L-1和NlDNM1L-2在褐飞虱卵巢中的表达和定位。这些结果为深入研究NlDNM1L-1和NlDNM1L-2在褐飞虱中的功能奠定了基础。
[Abstract]:[objective] to study the biological function of two dynamin-1-like genes, NlDNM1L-1 and NlDNM1L-2, in brown planthopper. [methods] the two genes of dynamin-1-like were amplified by polymerase chain reaction and their bioinformatics were analyzed. The relative expression of these two genes in different tissues and stages of development of brown planthopper was detected by fluorescence quantitative PCR technique. Prokaryotic expression vector was constructed to induce the expression of recombinant protein in Rosetta. The purified protein was purified by Ni column affinity chromatography, and the purified protein was immunized to New Zealand white rabbits. The polyclonal antibody was obtained. The antibody titer was detected by ELISA and the specificity of the antibody was detected by Western blotting. The expression and localization of the target protein in the ovaries of brown planthopper were observed by immunofluorescence assay. [results] two dynamin-1-like genes were cloned and identified, named NlDNM1L-1 and NlDNM1L-2(Gen Bank accession number KY082900 KY082901, respectively. Phylogenetic tree showed that NlDNM1L-1 and NlD NM1L-2 were conserved in Hemiptera. Protein structure prediction and gene expression analysis showed that NlDNM1L-1 and NlDNM1L-2 might have different functions in brown planthopper. The results of Elisa and Western blotting showed that the prepared polyclonal antibodies of NlDNM1L-1 and NlDNM1L-2 had high titer and good specificity. The results of immunofluorescence assay showed that NlDNM1L-1 and NlDNM1L-2 were widely expressed in follicular cells of brown planthopper ovary, which may be related to the development and maturation of ovaries of brown planthopper. They may also be related to the invasion of brown planthopper ovaries by yeast symbiotic bacteria. [conclusion] NlDNM1L-1 and NlDNM1L-2 gene sequences were obtained, their basic biological information was understood, and their space-time expression characteristics were elucidated, and their polyclonal antibodies were successfully prepared. The expression and localization of NlDNM1L-1 and NlDNM1L-2 in the ovaries of brown planthopper were preliminarily understood. These results lay a foundation for further study of the functions of NlDNM1L-1 and NlDNM1L-2 in brown planthopper.
【作者单位】： 中国计量大学生命科学学院浙江省生物计量及检验检疫技术重点实验室;
【基金】：国家自然科学基金资助项目(31501632) 浙江省自然科学基金资助项目(LQ14C140006) 国家973计划资助项目(2012CB114105)
【分类号】：S435.112.3
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