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褐飞虱锚定蛋白基因NlGPI的克隆及功能分析
Cloning and functional analysis of the brown planthopper, Nilaparvata lugens (St?l) anchor protein gene (NlGPI)
熊振泽;张 诺;宋 阳;申屠旭萍;俞晓平
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DOI:10.7679/j.issn.2095-1353.2022.106
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,杭州 310018;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,杭州 310018
中文关键词:褐飞虱;NlGPI;类酵母共生菌;基因功能
英文关键词:brown planthopper; NlGPI; yeast-like symbionts; gene function
中文摘要:
【目的】 为探明褐飞虱Nilaparvata lugens锚定蛋白基因NlGPI的表达模式和生物学功能。【方法】 基于褐飞虱转录组数据,利用PCR技术克隆NlGPI全长cDNA序列;利用生物信息学手段分析其核酸和蛋白质序列特征;通过qRT-PCR技术检测其时空表达规律;利用RNAi技术探明NlGPI的生物学功能。【结果】 克隆获得了褐飞虱NlGPI cDNA序列全长(GenBank登录号: XM_022342741.2),NlGPI的ORF共有813个核苷酸,编码 270个氨基酸,预测的分子量约为27 kD,预测的等电点为 4.84。该基因具有信号肽(Sec/SPI),且信号肽的剪切位点位于第19号和第20号氨基酸之间。该基因包含 1个具有GltG蛋白的结构域,共有 44 个磷酸化修饰位点。系统进化树分析表明,NlGPI与灰飞虱Laodelphax striatellus的亲缘关系最为接近,且在昆虫中的表达高度保守。qRT-PCR分析表明,NlGPI基因具有明显的时空特异性,NlGPI在卵期至初羽化基本不表达,羽化24 h后表达水平开始显著增加,羽化后72 h基因表达水平显著高于其它龄期。NlGPI在胸部、腹部、卵巢和肠道内的表达水平无显著差异(P>0.05),但均显著高于头部(P<0.05)。RNAi分析结果显示:NlGPI干扰后褐飞虱血淋巴中的类酵母共生菌(Yeast-like symbiont, YLS)数量显著下降,在注射24、48和72 h后,干扰组YLS数量比对照组分别减少了72.4%、68.2%和72.3%。NlGPI干扰后褐飞虱死亡率显著上升,干扰组到第10天褐飞虱全部死亡,而对照组则在第16天全部死亡。NlGPI干扰后的产卵量及孵化率均显著下降,对照组的褐飞虱产卵量最高为300 颗,最低为80 颗,未孵化的卵数量小于18 颗,平均孵化率大于90%;而处理组褐飞虱的产卵量最高为56 颗,未孵化的卵数量大于15 颗,平均孵化率小于20%。【结论】 NlGPI对褐飞虱体内YLS释放至血淋巴这一过程紧密相关,并在褐飞虱的生长发育及繁殖过程中发挥重要作用,可作为褐飞虱防控的潜在靶标。
英文摘要:
[Objectives]  To elucidate the expression pattern and biological function of anchor protein gene (NlGPI) from the brown planthopper, Nilaparvata lugens (Stål). [Methods]  Based on transcriptome data, the full-length cDNA of the NlGPI gene was cloned with PCR and its nucleic acid and protein sequence characteristics were analyzed with bioinformatics. qRT-PCR was then used to detect the gene’s temporal and spatial expression patterns and its biological functions were determined using RNAi. [Results]  The full-length cDNA sequence of NlGPI (GenBank Accession Number: XM_022342741.2) was cloned and obtained. The ORF contained 813 nucleotides encoding 270 amino acids with a predicted molecular weight of about 27 kD and a predicted isoelectric point of 4.84. The gene has a signal peptide (Sec/SPI), the spline site of which is located between amino acids 19 and 20. This gene contains a GltG protein domain, with a total of 44 phosphorylation modification sites. A phylogenetic tree indicates that NlGPI is most closely related to the homologous gene of Laodelphax striatellus, and that anchor protein genes are highly conserved in insects. qRT-PCR indicates that the NlGPI gene displays obvious temporal and spatial specificity. The gene NlGPI was hardly expressed from the egg stage to the beginning of emergence, its expression level only increasing significantly in 1-day-old female adults. Maximum expression level was recorded in 72 h-old female adults, which was significantly higher than that in other age groups. There was no significant difference of NlGPI expression in the thorax, abdomen, ovary and intestine, but the expression level in these organs was significantly higher than in the head. The number of YLS in the interference group 24, 48 and 72 h after RNAi treatment decreased by 72.4%, 68.2% and 72.3%, respectively, relative to the control group. NlGPI knockdown significantly increased N. lugens mortality; all N. lugens in the interference group died on the 10th day after treatment, while those in the control group survived until the 16th day. Knockdown also significantly decreased the number of eggs laid and the hatching rate. In the control group, the number of eggs laid ranged from 80 to 300 and the average hatching rate was > 90%. However, in the treatment group, the maximum number of eggs laid was 56 and the average hatching rate was < 20%. [Conclusion] NlGPI is closely related to the release of YLS from the fat body to the hemolymph and plays an important role in the growth, development and reproduction of the brown planthopper. The NlGPI gene is therefore a potential target for controlling this pest.
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