褐飞虱两个dynamin-1-like基因的克隆、多克隆抗体制备及 表达定位

doi:10.16819/j.1001-7216.2017.6165 345

摘要/Abstract

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(GenBank登录号分别为KY082900、KY082901)。系统进化树表明,NlDNM1L-1和NlDNM1L-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。

关键词: 褐飞虱, Dynamin-1-like, 基因克隆, 表达模式, 原核表达, 多克隆抗体, 表达定位

Abstract:

【Objective】To study the biological function of two dynamin-1-like genes, NlDNM1L-1 and NlDNM1L-2 in Nilaparvata lugens,【Method】 two dynamin-1-like genes were cloned from N. lugens by polymerase chain reaction (PCR), and their bioinformatics analysis was conducted. Using fluorescence quantitative real-time PCR technology, the relative expression levels of NlDNM1L-1 and NlDNM1L-2 in different tissues and at different developmental stages of N. lugens were detected. The expression vectors of the two genes were constructed and recombinant proteins were expressed in Escherichia coli Rosetta. The expressed recombinant proteins were purified by Ni-NTA agarose gel affinity system and then injected into New Zealand white rabbit to generate polyclonal antibodies. The titer of the antibodies was monitored by ELISA, and the immune specificity was determined by Western blotting hybridization. Using above antibodies, the localization of target proteins in ovary was detected by immunofluorescence. 【Result】The two dynamin-1-like genes were cloned, and their GenBank accession numbers were KY082900 and KY082901, respectively. The phylogenetic tree showed that NlDNM1L-1 and NlDNM1L-2 were conserved in Hemipterans. The results of protein structure prediction and space-time expression pattern analysis indicated that NlDNM1L-1 and NlDNM1L-2 might play different roles in N. lugens. The ELISA and Western blotting results showed that the polyclonal antibodies had high titer and good specificity. Immunofluorescence showed that NlDNM1L-1 and NlDNM1L-2 were widely distributed in the follicle cells of N. lugens ovary, indicating that they may be related to the development and maturity of N. lugens ovary. NlDNM1L-1 and NlDNM1L-2 may also be associated with the invasion of the yeast-like symbionts to N. lugens ovary. 【Conclusion】 The DNA sequences, bioinformatics and expression pattern of NlDNM1L-1 and NlDNM1L-2 were clarified. And the polyclonal antibodies of NlDNM1L-1 and NlDNM1L-2 were obtained and their localizations in the ovary have preliminarily been understood. These results lay a foundation for further biological function investigation of NlDNM1L-1 and NlDNM1L-2 in N. lugens.

Key words: Nilaparvata lugens, dynamin-1-like, gene cloning, expression pattern, prokaryotic expression, polyclonal antibody, localization

赵晨星, 俞叶微, 许益鹏, 俞晓平. 褐飞虱两个dynamin-1-like基因的克隆、多克隆抗体制备及表达定位[J]. 中国水稻科学, 2017, 31(4): 345-354.

Chenxing ZHAO, Yewei YU, Yipeng XU, Xiaoping YU. Gene Cloning, Polyclonal Antibody Preparation and Expression Localization of Two dynamin-1-like Genes from Nilaparvata lugens (Hemiptera: Delphacidae)[J]. Chinese Journal OF Rice Science, 2017, 31(4): 345-354.

图/表 9

表1 基因克隆、原核表达引物碱基序列

Table 1 Primer sequences used for gene clone and prokaryotic expression.

引物名称 Primer name	正向引物(5′-3′) Forward sequence(5′-3′)	反向引物(5′-3′) Reverse sequence(5′-3′)
基因克隆 Gene clone
NlDNM1L-1	GGCTTTGTTGGTGCTTTCA	CGAACCGGAATAGCCTTCT
NlDNM1L-2 原核表达 Prokaryotic expression NlDNM1L-1 NlDNM1L-2 定量PCR qPCR qNlDNM1L-1 qNlDNM1L-2 qNl18SrRNA	CTTTCTTGCGATTGTCACGC GGATCCATGGAAGCTCTTATACCAGTTACCA GAATTCATGGAAGCCTTGATACCAGTGATAA CGGCACCGATATTATACAGCTTC CAAAGCAGCGACAACGAATC GTAACCCGCTGAACCTCC	GGCCCAAAAGGAATCAACTA AAGCTTCTACCACAGATGTATTTCTCTGATCT AAGCTTCTACCACAGGTGAGTCTCGCGAACT AAGTTGAAGAACCAAGGGACACC CTCAGGCAGAAGATTGATGGG GTCCGAAGACCTCACTAAATCA

图1 NlDNM1L-1和NlDNM1L-2氨基酸序列比对

Fig. 1. Amino acid sequence alignments of NlDNM1L-1 and NlDNM1L-2.

图2 NlDNM1L-1(A)和NlDNM1L-2(B)蛋白三级结构预测

Fig. 2. Tertiary structure of NlDNM1L-1(A) and NlDNM1L-2(B) protein.

图3 NlDNM1L-1和NlDNM1L-2氨基酸序列的系统进化分析

Fig. 3. Phylogenetic analysis of NlDNM1L-1 and NlDNM1L-2 by the neighbor-joining method.

图4 NlDNM1L-1和NlDNM1L-2在褐飞虱的不同组织(A、B)和不同发育时期(C、D)的表达

Fig. 4. Expression of NlDNM1L-1 and NlDNM1L-2 in different tissues(A, B) and at different developmental stages(C, D).

图5 克隆载体的构建(A)和原核表达载体的构建(B、C)

Fig. 5. Construction of cloning vector(A) and prokaryotic expression vector(B and C).

图6 重组蛋白在E.coli Rosetta中表达的SDS-PAGE(A)分析及重组蛋白的纯化(B、C)

Fig. 6. Expression analysis(A) and purification(B and C) of expressed NlDNM1L-1 and NlDNM1L-2 in E. coli Rosetta by SDS-PAGE.

图7 Western blotting检验多克隆抗体

Fig. 7. Western blotting analysis of polycolonal antibodies.

图8 免疫荧光检测NlDNM1L-1和NlDNM1L-2在褐飞虱卵巢中的表达

Fig. 8. Expression analysis of NlDNM1L-1 and NlDNM1L-2 in N. lugens ovary by immunofluorescence.

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