本文選題：褐飛虱 + Dynamin--like　； 參考：《中國水稻科學》2017年04期

【摘要】：【目的】研究dynamin-1-like的兩個基因NlDNM1L-1和NlDNM1L-2在褐飛虱中的生物學功能�！痉椒ā客ㄟ^聚合酶鏈式反應擴增褐飛虱dynamin-1-like的兩個基因,并對其生物信息學進行分析。通過熒光定量PCR技術(qPCR)檢測了這兩個基因在褐飛虱不同組織和不同發(fā)育階段中的相對表達量。構建原核表達載體,在表達菌株Rosetta中誘導重組目的蛋白的表達。通過Ni柱親和層析純化目的蛋白,并將純化得到的蛋白免疫新西蘭大白兔,得到多克隆抗體,并用ELISA檢測抗體效價,Western blotting檢測抗體特異性。利用上述抗體,通過免疫熒光實驗觀察目的蛋白在褐飛虱卵巢中的表達和定位。【結果】克隆并鑒定了兩個dynamin-1-like基因,分別命名為NlDNM1L-1和NlDNM1L-2(Gen Bank登錄號分別為KY082900、KY082901)。系統(tǒng)進化樹表明,NlDNM1L-1和NlD NM1L-2在半翅目昆蟲中較為保守。蛋白結構預測和基因時空表達分析說明,NlDNM1L-1和NlDNM1L-2在褐飛虱中可能有著不同的功能。ELISA和Western blotting結果表明,制備的NlDNM1L-1和NlDNM1L-2多克隆抗體效價高、特異性好。免疫熒光實驗結果顯示,NlDNM1L-1和NlDNM1L-2在褐飛虱卵巢濾泡細胞中普遍表達,可能與褐飛虱卵巢發(fā)育和成熟有關,它們也可能與類酵母共生菌入侵褐飛虱卵巢有關。【結論】獲得了NlDNM1L-1和NlDNM1L-2基因序列,了解了其基本的生物信息,并闡明了其時空表達特征;成功制備其多克隆抗體,并初步了解了NlDNM1L-1和NlDNM1L-2在褐飛虱卵巢中的表達和定位。這些結果為深入研究NlDNM1L-1和NlDNM1L-2在褐飛虱中的功能奠定了基礎。
[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.
【作者單位】： 中國計量大學生命科學學院浙江省生物計量及檢驗檢疫技術重點實驗室;
【基金】：國家自然科學基金資助項目(31501632) 浙江省自然科學基金資助項目(LQ14C140006) 國家973計劃資助項目(2012CB114105)
【分類號】：S435.112.3
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本文編號：1864730